changes in serum homocysteine levels … surgery in severe obesity by ... for helping me fix the...
TRANSCRIPT
CHANGES IN SERUM HOMOCYSTEINE LEVELS AFTER ROUX-EN-Y GASTRIC
BYPASS SURGERY IN SEVERE OBESITY
By
BRANDON JAY BLANK
A Thesis Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY
in Partial Fulfillment of the Requirements
for the Degree of
MASTER OF SCIENCE
in the Department of Health and Exercise Science
May 2009
Winston-Salem, North Carolina
Approved By:
Gary D. Miller, Ph.D., Advisor ______________________________
Examining Committee:
Peter H. Brubaker, Ph.D. ______________________________
Barbara J. Nicklas, Ph.D. ______________________________
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DEDICATION
To Mom and Dad- For raising me and putting me in the position to be here and write this. Without your
ceaseless prayers, support, and love through the years, I would probably be selling hot dogs on the corner, or quite possibly in jail. Thank you for all that you have done and
continue to do for me/us.
To Katie- For your unending love and support. I could not have done this without you behind me at every step. Your love has carried me when I did not have the strength to do it on my own. Thoughts of our future and little Bailey’s college education have pushed me through and encouraged me that it truly is worth it. You are the love of my life and I can’t wait to sit on the front porch in a rocking chair with you after my sight, hearing, and knees have
failed me.
God- For your mercy and grace. I do not deserve your love and blessings, yet here I am. None of this would be possible without your strong hand on my life and redemptive work on
the Cross.
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ACKNOWLEDGEMENTS
I would like to thank the following people for the various support, knowledge shared, acts of kindness, and fellowship they provided over the last two years:
Dr. Gary Miller – For being my trusted advisor that was always there to meet with me to provide advice and M+M’s! For always connecting me to the right people whenever I needed something or wanted a new experience.
Dr. Pete Brubaker – For serving on my committee despite being incredibly busy. For helping me study for my Exercise Specialist Exam and buying us bagels and coffee every now and then. It has been a pleasure being one of your students.
Dr. Barbara Nicklas – For serving on my committee and allowing me to use you're your laboratory to run my assays.
Karen Murphy – For helping me fix the numerous errors that I made while running my folate and B-12 assays. For having the heart of a helper and truly being a wonderful person to work with. Brian Moore- For making time to sit down and try to help me with my statistics. Not an easy task!
Dr. Tim Kute – For having the heart of a teacher and spending time with me in your lab. I never dreamed I would have had the opportunity to culture and analyze cancer cells during my time here!
Dr. Fernandez – For allowing me to observe one of your gastric bypass surgeries and for performing the surgery on my participants.
Jim Ross- For allowing me to continue to work at HELPS in the GXT lab two days a week while providing great advice and wisdom about exercise testing and ECG interpretation.
Dr. Muday and Heather – For allowing me to hang out in your lab and learn how to run leptin assays.
Jovita, Monica, and Dr. Messier – For allowing me to spend time working at the IDEA study. Ben, Joel, LoLo, Suz, Manders, and Liz – For providing me with wonderful friendship, fellowship, and memories. The six of you truly made my time here enjoyable and I will always look back fondly of our experiences together from intramurals to trying to get out of putting up the HELPS posters up at 5:45am! I look forward to many years of friendship and laughter!
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TABLE OF CONTENTS
Page
LIST OF FIGURES…………………………………………………………………...viii
LIST OF TABLES…………………………………………………………………..….ix
ABSTRACT…………………………………………………………………………..….x
LITERATURE REVIEW……………………………………………………………..…..1
Introduction……………………………………………………………………......1
Obesity…………………………………………………………………………….2
Definition………………………………………………………………….2
Epidemiology and Costs…………………………………………………..2
Comorbidities……………………………………………………………...3
Cardiovascular disease…………………………………………………………….5
Incidence and Mortality…………………………………………………...5
Costs……………………………………………………………………….6
Risk Factors……………………………………………………………….6
Homocysteine……………………………………………………………………..7
Biochemistry………………………………………………………………7
Methionine Cycle………………………………………………………….9
Remethylation……………………………………………………………..9
Transsulfuration………………………………………………………….10
Reference Ranges for Plasma Homocysteine……………………………11
Determinants of Plasma Homocysteine………………………………….12
v
Genetic Factors…………………………………………………..13
Physiological Determinants……………………………………...16
Drug and Disease Determinants………………………………….17
Life-Style Determinants………………………………………….18
Homocysteine and Cardiovascular Disease……………………………………...27
Homocystinuria and Cardiovascular Disease……………………………27
Hyperhomocysteinemia and Cardiovascular Disease……………………28
Mechanisms……………………………………………………………...31
Cytotoxicity………………………………………………………31
Inflammatory Response………………………………………….31
Oxidative Stress and Endothelial Function………………………32
Smooth Muscle and Collagen Proliferation……………………...33
Treatments for Obesity………………………………………………………….33
Weight Reduction Surgery………………………………………………36
Roux-En-Y Gastric Bypass Surgery……………………………………………..38
Surgery Description……………………………………………………...38
Weight Loss……………………………………………………………...39
Side Effects………………………………………………………………40
Vitamin Deficiencies…………………………………………………….42
Vitamin B-12…………………………………………………….42
Folic Acid………………………………………………………..43
Surgical Experience…………………………………………………...44
Health Benefits…………………………………………………………...45
vi
Mortality…………………………………………………………45
Diabetes…………………………………………………………..47
Hyperlipidemia……………………………………………..……47
Hypertension…………………………………………………….48
Sleep Apnea…………………………………………………..…48
Homocysteine………………………………………………..….49
Specific Aims of the Study………………………………………………………51
Hypotheses……………………………………………………………………….51
METHODS………………………………………………………………………………53
Recruitment and Eligibility Requirements………………………………………53
Informed Consent………………………………………………………………..54
Study Procedures and Data Collection…………………………………………..55
Nutritional Supplementation……………………………………………………..56
Homocysteine Measurement……………………………………………………..57
Folic Acid Measurement…………………………………………………………57
Vitamin B-12 Measurement……………………………………………………...58
Drop Outs………………………………………………………………………59
Statistical Analysis……………………………………………………………….59
RESULTS………………………………………………………………………………..61
Homocysteine……………………………………………………………………66
Serum Folic Acid……………………………………………………………..69
Serum Vitamin B-12…………………………………………………………….72
Dietary Results…………………………………………………………………..75
vii
Correlations………………………………………………………………………77
DISCUSSION……………………………………………………………………………82
REFERENCES…………………………………………………………………………..91
viii
LIST OF FIGURES
FIGURES PAGE 1 Homocysteine Metabolism………………………………………………………11 2 Weight at Baseline and Across 12 Months of Follow Up….……………………64 3 Percent Weight Lost Between Baseline and Follow-Up Visits………………….65 4 Mean Serum Homocysteine Levels…………………………………………..…67 5 Individual Serum Homocysteine Levels…………………………………………68 6 Mean Serum Folic Acid Levels…………………………………………………70 7 Individual Serum Folic Acid Levels………………………………………….71 8 Mean Serum Vitamin B-12 Levels………………………………………………73 9 Individual Serum Vitamin B-12 Levels………………………………………….74 10 Correlation Between Change in HCY and BMI Lost …………………………...79 11 Correlation Between Change in HCY and Change in B-12……………………..80 12 Correlation Between Change in HCY and Change in Folic Acid……………….81
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LIST OF TABLES
TABLES PAGE 1 Traditional Risk Factors for Cardiovascular disease………………………….6 2 Emerging Risk Factors for Cardiovascular Disease…………………………7 3 Patient Characteristics………………………………………………………..62 4 Dietary Intake ………………………………………………………………76 5 Pearson-Product Correlations………………………………………………78
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ABSTRACT
Homocysteine is a sulfur-containing amino acid that is formed after the
demethylation of methionine. High levels of plasma homocysteine have been proposed as
a major and independent risk factor for cardiovascular disease. It is well known that
obesity is also a modifiable and independent risk factor for cardiovascular disease.
Extensive research has shown that weight loss produced by both traditional methods
(dietary restriction/exercise) and gastric bypass surgery can improve several well known
cardiovascular risk factors. As the rates of morbid obesity have been rapidly growing, the
number of gastric bypass surgeries has also drastically increased, with Roux-en-Y gastric
bypass (RNYGBP) surgery being the most common. While it is relatively well known
that gastric bypass and weight loss improve many other risk factors for cardiovascular
disease, the literature has been very limited regarding the effect of gastric bypass surgery
on plasma homocysteine concentrations. The purpose of this study is to examine the
effect of this surgery on the emerging cardiovascular disease risk factor of high plasma
homocysteine. Participants were recruited from patients scheduled to undergo gastric
bypass surgery at the Wake Forest University Baptist Medical Center General Surgery
Clinic. Men and women were eligible for the study if they had a BMI ≥ 40.0 kg/m2 or ≥
35.0 kg/m2 with an obesity related comorbidity, such as hypertension, dyslipidemia, or
diabetes. Participants (n=19 female; age=45.7 (±8.8) years; BMI=54.4 (±7.2) kg/m2 had
blood drawn prior to, and at 3 weeks, 3 months, 6 months, and 12 months post-surgery.
Blood serum was analyzed for homocysteine, as well as vitamins B-12 and folate because
these water-soluble B vitamins are determinants of serum homocysteine levels. One-way
repeated measure ANOVA determined effect of the surgery on homocysteine over time.
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Weight loss was -7.8 (±1.5)% at 3 weeks, -17.7 (±2.7)% at 3 months, -26.0 (±4.0)% at 6
months, and -33.8 (±7.1)% at 12 months. Serum folate and vitamin B-12 did not
significantly change from baseline to 12 months. Serum homocysteine levels were 10.5
(±3.6) μM/L at baseline, 11.8(±3.9) μM/L at 3 weeks, 11.4(±3.9) μM/L at 3 months,
11.7(±2.9) μM/L at 6 months, and 10.4 (±2.4) μM/L at 12 months. Serum homocysteine
levels did not significantly change during the study. At baseline and throughout the
course of the study, 95% of the participants had normal homocysteine levels. Change in
homocysteine at twelve months was significantly negatively correlated to change in BMI
as well as change in weight between baseline values and other visits. Results indicate that
when vitamin status is controlled, Roux-en-Y gastric bypass surgery will not significantly
alter homocysteine levels in morbidly obese patients.
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LITERATURE REVIEW
INTRODUCTION
Homocysteine is a sulfur-containing amino acid that is formed after the
demethylation of methionine.99, 100, 138 High levels of plasma homocysteine have been
proposed as a major and independent risk factor for cardiovascular disease.54, 99-101, 135, 138
It is well known that obesity is also a modifiable and independent risk factor for
cardiovascular disease169. Extensive research has shown that mass loss produced by both
traditional methods (dietary restriction/exercise) 6, 95, 98 and gastric bypass surgery 94, 168,
217 can improve several well known cardiovascular risk factors. As the rates of morbid
obesity have been rapidly growing, the use of gastric bypass surgeries to promote mass
loss has also drastically increased, with Roux-en-Y gastric bypass (RNYGB) surgery
being the most common.5, 48, 63, 180 While it is relatively well known that gastric bypass
and mass loss improve many other risk factors for cardiovascular disease, the literature
has been very limited regarding the effect of gastric bypass surgery on plasma
homocysteine concentrations.24, 53, 94, 99, 118 Thus, it is of interest to examine if this surgery
alters levels of the emerging cardiovascular disease risk factor of high plasma
homocysteine.
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OBESITY
Definition
Obesity is commonly defined as an abnormal or excessive amount of body fat that
leads to impaired health.204 While there are many ways to establish the presence of
obesity, the most commonly used definition is body mass index (BMI). BMI is the ratio
of mass (kg) to height squared (m²). Typically, a high BMI will indicate a high amount of
body fat, as well as increased health risks.79 While the level of adiposity shows a high
inter-individual variation for a given BMI, overall BMI has been shown to be a relatively
accurate predictor of adiposity on the population level.19, 79 A person with a BMI over 30
kg/m2 is considered obese. There are three classes of obesity. Class 1 obesity is a BMI
between 30.0 and 34.9 kg/m2; class 2 is between 35.0 kg/m2 and 39.9 kg/m2; and class 3
is greater than 40.0 kg/m2. Class 3 obesity is often referred to as “Morbid Obesity”.2 In
recent years, increased attention has been paid to the regional location and distribution of
the fat stores. Research has shown that abdominal obesity might have a greater bearing
on poor health than BMI alone or fat located subcutaneously.50, 79, 102, 105
Epidemiology and Costs
In the last two decades, the prevalence of obesity has increased dramatically in
America. Since 1985, the obesity rate in America has doubled among adults, and tripled
among children. According to the Center for Disease Control the prevalence rate is
currently over 26% in the obese category and 60% of Americans are either overweight or
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obese (BMI ≥ 25 kg/m2).35 NHANES studies have predicted an even higher prevalence
rate at 66.3% overweight and 32.2% obese163. In addition, NHANES studies predict that
4.8% of the population is morbidly obese with a BMI over 40.0 kg/m2.163 While these
statistics are alarming, the rate at which they are increasing is even more so. Just twenty
years ago the obesity rate was below 15% with only one in 200 being morbidly obese vs.
the current rate of one in three that are obese with one in twenty classified as morbidly
obese. The rates have been projected such that by 2030, close to 90% of American adults
will be overweight, and 51% will be obese.211
With such a rapidly increasing prevalence, obesity undoubtedly has a major
impact on American economy and the health of its citizens. It has been estimated that
obesity directly cost well over 100 billion dollars per year in America.87, 218 This number
does not take into account the fact that obesity is a risk factor for several other diseases
costing billions of dollars a year.87, 218 Health professionals project that this increase will
raise annual health care costs to close to $900 billion dollars.211
Comorbidities
It is very well known that obesity has several different repercussions for an
individual’s health. Obesity has been shown to substantially raise the risk of hypertension,
dyslipidemia, type 2 diabetes, coronary heart disease, stroke, gallbladder disease,
osteoarthritis, sleep apnea and respiratory problems, and endometrial, breast, prostate,
and colon cancers.2, 32, 106, 148, 204, 211, 218 An increase of any and all of these conditions
affects the mortality of this population. In a large analysis of cohort studies, Allison et al.
estimated that roughly 300,000 American deaths are attributable to obesity each year
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(1991).6 It has been shown that a forty-year-old female and male non-smoker will lose
3.3 and 3.1 years of life, respectively, if they are overweight. Those numbers increase to
7.1 and 5.8 years respectively if they are obese.144 In another study, it was shown that
subjects in the highest BMI indexes had a relative risk of death of 2.58 and 2.00 (men and
women respectively) compared to those with a ‘normal’ BMI. In this same study, a high
BMI was most predictive of death caused by cardiovascular disease which had a relative
risk of 2.90 in men.32 Willett et al showed that women who have a BMI of 29 kg/m2 and
above had a relative risk of 3.56 for developing coronary heart disease when compared to
women with a BMI of under 21 kg/m2.216 A recent study showed that an increase of
3kg/m² in BMI raises risk for thromboembolic stroke by 10% to 30%. 162
Obesity plays such a large role in the development of cardiovascular disease
mainly because it raises several of the well-known risk factors for the disease. Obesity
has been shown to increase blood pressure, diabetes, and LDL cholesterol while
decreasing HDL cholesterol, all of which have been significantly correlated with
cardiovascular disease.163 The effect that obesity has on plasma homocysteine levels is a
little more debatable. Very little research has examined this topic, and that which has
been done has been rather inconclusive. One group found that plasma homocysteine is
significantly associated with waist-to-hip ratio, but not BMI.107 Another group of
researchers conducted two separate studies that examined plasma homocysteine levels in
hypertensive and diabetic obese/non-obese subjects. In both studies plasma homocysteine
levels were significantly correlated to obesity.99, 100 In a dietary intervention using a
prepared meal plan, subjects with total plasma homocysteine levels of 10.8 μM/L lost an
average of 4.8 ±3.0 kg of body mass and their homocysteine was reduced by 1.5 ± 3.3
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µmol/L (p<0.01).118 This not only indicates that body mass (and therefore BMI) might be
correlated with plasma homocysteine levels, but it also suggests that mass loss might be a
viable treatment for high plasma homocysteine levels.
CARDIOVASCULAR DISEASE
Incidence and Mortality
It has been well documented that obesity raises the risk of developing
cardiovascular disease. While there are many forms of cardiovascular disease, when
lumped together, they are by far America’s number one killer. Roughly 2,400 Americans
die from cardiovascular disease every day. That is more deaths than diabetes, respiratory
disease, cancer, and accidents combined.131 Cardiovascular disease was the underlying
cause of death in roughly 36% of all deaths in 2004. However, cardiovascular disease is
mentioned in the cause of death in 57% of all deaths that year.131 The National Center for
Health Statistics predicted that if all forms of cardiovascular disease were eliminated, the
life expectancy in America would rise by seven years.9 While the death rates of
cardiovascular disease are actually decreasing131, the actual prevalence of the disease is
not. Roughly 15% of men between the age of 55 and 64 have cardiovascular disease,
while that number jumps to 35%, 52%, and 69% for the ages between 65-74, 75-84, and
85-94 respectively.82 While people are living longer and life expectancy is going up, this
suggests that more and more people will live with cardiovascular disease. In 2005, it was
estimated that close to 80 million Americans had some form of cardiovascular disease.
Close to 40 million of those were over the age of sixty. Seventy-three million people
either had hypertension, or were being treated for it. Sixteen million people had coronary
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heart disease, eight million of whom had a myocardial infarction, and nine million who
had angina. Of the eighty million cardiovascular disease patients, five million people had
heart failure. An additional six million had a stroke.147
Costs
With so many people affected by cardiovascular disease, the economic burden is
very high. In 2005, close to seven million inpatient procedures were performed on
cardiovascular disease patients.47 The estimated direct and indirect cost associated with
cardiovascular disease is 448.5 billion dollars for 2008. 167
Risk Factors
There has been an abundant amount of research done to determine the causes and
risk factors for cardiovascular disease. Through this research, a well-established list of
risk factors has been created. These risk factors are commonly referred to as the
‘traditional risk factors.’56
TABLE 1. Traditional Risk Factors for Cardiovascular Disease
Tobacco Smoking Obesity
Hypercholesterolemia Insulin Resistance Syndrome
Elevated Plasma LDL cholesterol Sedentary Lifestyle
Low HDL cholesterol Age
Hypertriglyceridemia Menopause
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Hypertension Male Sex
Diabetes Mellitus Family History
In addition to the traditional risk factors, there is large list of ‘nontraditional’ or
‘emerging’ risk factors that have been receiving a lot of attention recently. 56
TABLE 2. Emerging Risk Factors for Cardiovascular Disease
Elevated plasma lipoprotein A165 ACE gene polymorphism141
Elevated plasma lipoprotein B175 Increased oxidation of LDL179
Small dense LDL14 Mutations affecting platelet activation41
Paraoxonase deficiency111 Elevated plasma fibrinogen83
Infectious agents71 Hyperhomocysteinemia 55
Elevated plasma homocysteine levels have been shown in dozens of studies to be
a strong, independent risk factor for cardiovascular disease. While the nature and
mechanisms of the relationship is still debatable, there is ample evidence to provoke
further study into plasma homocysteine and its interaction with other cardiovascular
disease risk factors.
HOMOCYSTEINE
Biochemistry:
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Homocysteine is a sulfur-containing amino acid that is formed during the
demethylation of dietary methionine. 99, 100, 138 There are several forms of homocysteine.
Free reduced homocysteine accounts for about 2% of the total plasma homocysteine
(tHcy) levels in healthy individuals. In homocystinurics (extremely high total plasma
homocysteine) free reduced homocysteine may be as high as 20% of the total.
Homocysteine can oxidize with itself to create homocysteine. This accounts for roughly
5-10% of tHcy. Homocysteine can also oxidize with cysteine to form homocysteine-
cysteine mixed disulfide (also 5-10% of tHcy). The most common (80-90%) form of
plasma homocysteine is protein bound homocysteine. Albumin is thought to be the most
common protein carrier.92 Unless otherwise mentioned, a reference to plasma
homocysteine is typically referring to total plasma homocysteine.
There are very scarce levels of homocysteine in food sources, so almost all of it is
derived as an intermediate in the methionine cycle. 92 Methionine is an essential amino
acid that is found in the highest quantities in animal products such as eggs, meat, fish,
and milk at levels around 3 g/10 0g of protein. Methionine is also found in most plant
sources such as fruit, vegetables, nuts, and cereals at levels around 1 g/100 g of protein.
The exceptions to plant sources are peaches and grapes which have a content of 3.6 g/100
g, and Brazil nuts which have the highest content at 5.6 g/100 g. Consequently, a meal
very high in protein and methionine (over 50 grams) can elevate plasma homocysteine
levels. However, it has been shown that meals containing moderate levels of protein (15-
18 grams) do not have an effect on plasma homocysteine levels.76 It is estimated in
healthy people that around 1.32 mmol of homocysteine is exported from cells to the
blood each day, but only 0.006 mmol is secreted in the urine. This suggests that the body
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is able to metabolize a moderate amount of homocysteine through the methionine cycle
and only in rare circumstances when some part of the metabolism process is not working
correctly will homocysteine be excreted in urine.92
Methionine Cycle
The methionine cycle is shown in Figure 1. Dietary methionine is first introduced
in the cycle by the enzyme methionine adenosyltansferase (MAT). MAT catalyzes the
conversion of methionine into energy rich S-adenosylmethionine (SAM).
Methyltransferases then use SAM as the substrate for methyl-group transfers to a number
of different acceptors. Some processes that use methyl groups are synthesis of
phospholipids, proteins, DNA, RNA, creatine, and epinephrine creation, as well as gene
expression.92 After SAM donates its methyl group to its acceptor, S-
Adenosylhomocysteine (SAH) is formed. SAH is then hydrolyzed to create homocysteine
and adenosine by the enzyme SAH hydrolase. This is a reversible reaction; however, it is
undesirable as it might lead to excess SAH accumulation. SAH is a strong end-product
inhibitor and will impair methyltransferase activity.92
At this point, homocysteine serves as a branch point in the methionine cycle. It
will either undergo remethylation or transsulfuration.92 It has been shown that on a
normal diet, roughly 50% of the homocysteine goes through remethylation, and the other
50% goes through transsulfuration. 213
Remethylation
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The methionine cycle is completed once homocysteine is remethylated into
methionine. The remethylation of homocysteine is done by a second, separate cycle
called the folate cycle. Methionine synthase (MS) uses 5-methyltetrahydrofolate as the
methyl donor group and methylcobalamin (a coenzyme of vitamin B12) to convert
homocysteine back into methionine.17, 92 It appears that all tissue types have the ability to
remethylate homocysteine using this pathway.17, 58-62, 92
Transsfulfuration
The transsulfuration pathway uses the enzyme cystathionine synthase (CBS) and
vitamin B6 as a co-factor to irreversibly convert homocysteine into cystathionine.
Cystathionine is converted into the amino acid cysteine. Cysteine is used in a variety of
metabolic pathways or converted into inorganic sulfate and excreted in urine. 92, 213 It
appears that the transsulfuration pathway might be limited to certain cells however. For
example, it has been shown that there is very little CBS activity in cardiovascular
endothelial cells in both rats and humans.59, 92, 199, 210 Finkelstein and colleagues found
that CBS activity is limited to the liver, kidney, pancreas, adipose, brain, and possibly
intestinal mucosa. 59 The capacity of the transsulfuration pathway appears to be limited.
If the remethylation pathway is impaired for any reason, the transsulfuration pathway is
not able to handle the increased plasma homocysteine. 92 Any type of error in the
transsulfuration pathway will typically lead to high levels of homocysteine as well as
methionine. An error in the remethylation pathway will typically only raise homocysteine
levels while methionine levels remain normal or even low. 164
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FIGURE 1. Homocysteine Metabolism
Derived from Nygard, 1999138
Reference Ranges for Plasma Homocysteine
While specific standards have not been unanimously agreed upon, most literature
describes a normal plasma homocysteine level somewhere in between 5 and 15 μM/L. 8,
75, 142, 152, 191, 192, 197 However; most studies that exhibit this reference range are positively
skewed. 192 According to an analysis done on the Physicians Health Study, the risk for
myocardial infarction increased 3.4 fold when the plasma homocysteine level was in the
tailed part of the distribution of the curve.176 This suggests that it may be inappropriate to
include the skewed part of the curve in a ‘normal’ reference range for healthy people. 192
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Reference ranges that are generated using B-12 and folic acid vitamin supplementation,
which is a major determinant of plasma homocysteine levels (to be discussed later), lose
most of their skewness, suggesting that most cases of high homocysteine can be
attributed to a shortage of vitamin B-12 and folic acid. The studies that use this
supplementation show that a better reference range for tHcy levels is closer to 5-12 μM/L.
75, 108, 152, 191 Other studies have suggested upper levels as low as 11.7142, 11.4166, and 10.5
μM/L.113
Plasma homocysteine levels above the normal reference limit, whether that is 12
or 15 μM/L, but below 30 μM/L are given the definition moderate
hyperhomocysteinemia (also known as homocysteinemia). Intermediate
hyperhomocysteinemia is a plasma level between 30–100 μM/L. Acute
hyperhomocysteinemia is any plasma level >100 μM/L.8 When homocysteine reaches
levels this high, it is common that a large amount of homocysteine will be excreted in the
urine. This condition is called homocystinuria. Homocystinurics have been known to
have plasma homocysteine levels as high as 500 μM/L. 8
There are many determinants of plasma homocysteine levels, and it has been
proposed that separate reference ranges might need to be created for different populations.
192 For example, if a prepubescent child had a plasma homocysteine level of 12 μM/L,
that would be considered high because the average for that age is 5 μM/L.198
Determinants of Plasma Homocysteine
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There are considered five different categories of homocysteine determinants.
Genetic, physiological, diseases, drug, and life-style factors all influence total plasma
homocysteine levels. 198
Genetic Factors
Of all the determinants, genetic mutations have the largest capacity to affect
plasma homocysteine levels. There are many steps in methionine and homocysteine
metabolism, and a defect at any point can lead to hyperhomocysteinemia or
homocystinuria.
Cystathionine β Synthase (CBS) Deficiency
In the early 1960s, while searching for metabolic abnormalities as causes of
mental retardation, researchers observed that some patients were secreting a large amount
of homocysteine in their urine. These researchers found that the defect was the absence or
diminished activity of cystathionine synthase.213 As described above, CBS is responsible
for the irreversible degradation of homocysteine. Without CBS, the entire transsulfuration
pathway of the methionine cycle is shut off. Since the late 1960s, it has been well
researched and shown that homozygous deficiency of CBS is associated with
homocystinuria and precocious vascular disease.74 The effect heterozygosity for CBS
deficiency has on plasma homocysteine is not as clear. Fasting plasma homocysteine
levels in these individuals is typical normal or slightly elevated. After a methionine load,
however, plasma homocysteine will raise beyond normal levels. 27, 198
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Elevated plasma homocysteine levels due to CBS deficiency can be treated in
roughly 50% of patients with pyridoxine.129 In patients non-responsive to pyridoxine, oral
betaine is normally effective at lowering plasma homocysteine levels.215
MTHFR Deficiency
As described above, 5-methyltetrahydrofolate is the methyl donor to convert
homocysteine back into methionine. The enzyme methylenetetrahydrofolate reductase
(MTHFR) is required for this demethylation to occur. Severe deficiency of MTHFR will
result in homocystinuria, while milder deficiencies of the enzyme are only associated
with mild to moderate hyperhomocysteinemia.164 These deficiencies are most often the
result of a C677T polymorphism of the enzyme. Homozygosity for this condition will
impair folic acid status and provoke hyperhomocysteinemia.198 However, there have been
eighteen other rare MTHFR mutations identified. The C677T polymorphism is the only
one that has consistently shown to influence plasma homocysteine levels. In North
America, it has been shown that 11%-15% of the population is homozygous for C677T
mutation.67, 164 As mentioned previously, this specific mutation will interfere with folic
acid metabolism and can be expected to lower plasma folic acid levels. This decrease in
folic acid will in turn raise plasma homocysteine levels to a mild or moderate
hyperhomocysteinemia level. Normally, the folic acid levels in these individuals are not
considered in the deficient range, but rather in the low end of the ‘normal range.’164 There
have been several studies examining the relationship between the C677T mutation and
cardiovascular disease. These studies have shown that there is a significant correlation
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between the mutation and coronary artery disease 128, cerebral infarction 127, and venous
thrombosis.13, 116
In contrast, there have been studies that have shown no correlation between
C677T mutation and cardiovascular disease.96, 97, 109, 117 These discrepancies might be
explained due to several of the studies not assessing folic acid status or having a large
enough of a sample. 164
Despite the discrepancies, a meta-analysis concluded that the mutation of
MTHFR was a modest yet still significant risk factor for cardiovascular disease.97
Treatment for this mutation has proven to be relatively easy. Since folic acid status is
what decreased levels of MTHFR, it has been found that folic acid supplementation in
these patients is an effective way to overcome the effect of the mutation and restoring
normal plasma homocysteine levels. 164
MS Deficiency
As described previously, methionine synthase (MS) is the other enzyme crucial to
the remethylation of homocysteine into methionine.17, 92 Methionine synthase is found in
practically all mammalian tissues. MS is the enzyme responsible for demethylating
methyltatrahydrofolic acid. MS is dependent on a cofactor, methylcobalamin, which is
derived from vitamin B-12. Severe mutations of MS are correlated with severe
hyperhomocysteinemia and homocystinuria. There have not been any polymorphisms
found that lead to only moderate hyperhomocysteinemia. Deficiencies of methionine
synthase result most commonly from defects in the enzyme itself, or indirectly by
impaired cobalamin transport proteins. 17, 134, 198
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Physiological Determinants
Plasma homocysteine levels increase throughout life in both men and women. As
mentioned previously, the average total plasma homocysteine level in prepubescent
children is 5 μM/L for both boys and girls.198 During puberty, plasma homocysteine
levels will typically increase to 6-7 μM/L. This change is more pronounced in boys than
it is in girls.187, 198 The characteristically skewed distribution also takes form during this
stage in life.187 After puberty, plasma homocysteine increases 3-5 μM/L.198 Data from
NHANES III shows that plasma homocysteine is 40% (3.6 μM/L), 18% (1.6 μM/L), and
2.2% (0.2 μM/L) higher in adults over seventy years old, between fifty and seventy years
old, and between thirty and fifty years old, than it was in subjects younger than thirty,
respectively. 68 The mechanisms that lead to this age-related increase in plasma
homocysteine are not well understood, but it is believed that impaired renal function is
involved. 155, 56, 79 It has also been proposed that this increase with age might be due to
diminished cystathionine β Synthase activity. 70
In adulthood, plasma homocysteine levels are typically 1-2 μM/L higher in males
than in females (pre-menopausal). 198 Using data from NHANES III and the Hordaland
cohorts, it was shown that plasma homocysteine was 21.1% higher in men than in women
(1.9 μM/L). 68, 198 This disparity might be explained by differences in muscle mass,
hormones, and vitamin status. 155, 156 Plasma homocysteine levels in post-menopausal
women tend to be higher than pre-menopause and typically will resemble that of men the
same age.134, 198
- 17 -
Pregnancy has been shown to substantially lower plasma homocysteine (about a
50% reduction). During the first and second trimesters, plasma homocysteine will
decrease, and then stay relatively stable for the remainder of the pregnancy. Normally,
typical plasma homocysteine levels will be attained two to four days post-partum. 11
Maternal plasma homocysteine is inversely related to neonatal mass and gestational age
after delivery, which suggests that the decrease during pregnancy might be due to fetal
uptake of maternal plasma homocysteine.114, 198 Another hypothesis is that lower plasma
homocysteine levels are a physiological adaptation which supports placental circulation.
23, 198
Drug and Disease Determinants
There are a number of different drugs and diseases that can affect plasma
homocysteine levels. It appears that most of these drugs and conditions that effect plasma
homocysteine levels have a common variable: they interfere with the individual’s vitamin
status. 198 Vitamin status will be discussed more thoroughly in the lifestyle determinants
section, but it is well known that low levels of the vitamins associated with homocysteine
metabolism will affect plasma homocysteine levels accordingly.
Drugs
Methotrexate is used for cancer chemotherapy, leukemia, psoriasis, as well
rheumatoid arthritis patients. It is considered an antifolate drug, and in turn, can increase
plasma homocysteine levels up to 100 μM/L. 126, 151, 154, 157 Several other drugs also
- 18 -
interfere with folic acid status, such as phenytoin (anticonvulsant) and colestipol (bile
sequestrant). 198
Drugs that interfere with vitamin B-12 have also shown to increase plasma
homocysteine levels. Cholestyramine 45, histamine H2-receptor antagonists 66,
omeprazole 20, as well as metformin, have all been shown to interfere with B-12
absorption. However, increased plasma homocysteine levels have only been reported in
cholestyramine and metformin. These two drugs might also affect folic acid absorption. 34,
198 Many other drugs have the potential to alter plasma homocysteine levels; for a
complete list, see Ueland, 2000. 198
Diseases
Similar to drugs, any disease that negatively affects the absorption or function of
the vitamins crucial to homocysteine metabolism will cause plasma homocysteine to rise.
Some of these conditions are: renal failure 49, leukemia 151, and psoriasis. 154 Other
conditions that have been reported to affect plasma homocysteine levels are diabetes
(type I) 89, hypo/hyperthyroidism 132, and heart transplant patients. 22
Life-Style Determinants
While genetic disorders have the greatest potential to cause severe
hyperhomocysteinemia, those conditions are generally rare and account for only a
fraction of cases of elevated plasma homocysteine levels. With the exception of vitamin
B-12 deficiency, life style determinants do not have the same ability to drastically
increase plasma homocysteine levels. Instead, they are the most common cause of mild
- 19 -
to moderate hyperhomocysteinemia.198 As discussed previously, the general populations’
homocysteine distribution is positively skewed. It has been shown that the majority of the
individuals in the ‘tail’ of the distribution have some sort of vitamin deficiency.193 Indeed,
studies including vitamin supplementation exhibit a more symmetrical distribution. 152, 191
This evidence suggests that most cases of elevated plasma homocysteine levels are
directly related to poor nutrient status.
Protein and Methionine Consumption:
As discussed earlier, there is very little homocysteine in dietary sources.
Practically all homocysteine is derived directly through the methionine cycle. With that
in mind, it would seem obvious that a diet high in methionine would, in turn, increase
plasma homocysteine levels. Indeed, plasma homocysteine levels have been shown to
increase 14% eight hours after a protein (source of methionine) rich meal. 76 With that
said some studies show that homocysteine levels do not seem to be related to daily
dietary methionine or protein content. 10, 171 It appears that the methionine cycle is able to
adequately handle the typical diet, and although a large protein-rich meal will cause a
spike in plasma homocysteine, these changes are temporary, and levels return to normal
after an overnight fast. 203 There have been other reports that a high protein diet can
actually decrease fasting plasma homocysteine. It is hypothesized that a high daily
protein intake leads to more efficient catabolism of homocysteine through greater
activation of enzymes in the methionine cycle. In addition, high protein foods contain
other amino acids and vitamins that could influence homocysteine metabolism. 182 In
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animal studies, it has been shown that mainly the transsulfuration pathway is activated in
the animals on a high methionine diet. 59
Vitamin B-6 Status
Vitamin B-6 is used as a co-factor in the transsulfuration pathway and a
deficiency in this nutrient has the potential to increase levels of plasma homocysteine.
The metabolite effecter SAM is one of the ways the body controls the methionine cycle.
When methionine levels are high, SAM activates the transsulfuration pathway, and
inhibits the remethylation pathway. 92, 123 This limiting mechanism will dictate how much
vitamin B-6 is able to effect plasma homocysteine levels. Since B-6 is used only in the
transsulfuration pathway, as long as methionine, and thus SAM, levels are low, such as in
a fasting state, plasma homocysteine will not be affected. Since most of the time tHcy is
measured while fasting, vitamin B-6 deficiency does not seem to have an effect on
plasma homocysteine levels. However, if a vitamin B-6 deficient individual takes a
methionine load and then has tHcy measured, it will be dramatically elevated. 52, 123
Vitamin B-12 Status
Cobalamin, also known as vitamin B-12, is the main catalyst in the 5-
methtetrahydrofolate conversion of homocysteine into methionine. Without cobalamin,
the entire remethylation cycle essentially gets shut off.92 Vitamin B-12 deficiency is a
common cause of moderate to severe fasting hyperhomocysteinemia. 191, 193, 198 It is
estimated that a person deficient in cobalamin will have a fasting plasma homocysteine
level between 25 and 105, depending greatly on the severity of the deficiency, as well as
- 21 -
the presence of any of the other determinants discussed in this section. 198 Interestingly,
women typically have a higher cobalamin status than men. In a study that examined
NHANES III participants, women had an average of 463.6 pmol/L, while the men only
had 373.3 pmol/L.68 This difference might play a role in explaining why men have higher
plasma homocysteine levels than women. In this same study, they found that plasma
homocysteine and B-12 have a strong, negative association.68
Folic acid Status
Similar to cobalamin, folic acid is an essential nutrient in the remethylation
pathway of the methionine cycle. As discussed previously, tetrahydrofolate is the active
substrate in converting homocysteine back into methionine. Tetrahydrofolate is derived
from folic acid. Deficiencies in folic acid can lead to moderate to severe
hyperhomocysteinemia.92 While folic acid deficiency has the potential to lead to much
higher values of plasma homocysteine, under normal circumstances, it will only raise
plasma homocysteine levels to anywhere between 20 and 50 μM/L.198 In the NHANESIII
study, a negative linear association was found between folic acid status and plasma
homocysteine levels (P < 0.0001 for linear trend), demonstrating that folic acid plays a
significant role in plasma homocysteine levels. The multivariate-adjusted average plasma
homocysteine level was 34.8% (3.2 μM/L) higher in the participants with the lowest
plasma folic acid than those with the highest. Similar to B-12 status, it was found that
women have higher folic acid levels than men (by 2.3 nmol/L).68 Again, this might
explain some of the variance of homocysteine levels between men and women.
- 22 -
Smoking
Cigarette smoke, as well as plasma cotinine, has been shown to moderately raise
plasma homocysteine. Cotinine is a metabolite of nicotine and is directly related to
absorbed nicotine.21 Cotinine might be a better measure of smoking status because study
participants tend to underreport cigarette habits and smokers inhale cigarette smoke
differently.68 In NHANESIII, it was found that cotinine levels were positively correlated
to plasma homocysteine levels. People in the highest quartile of cotinine had significantly
higher plasma homocysteine than people in the lowest.68 In the massive (n=18,000)
Hordaland Homocysteine Study, one of the most important findings was that there is a
dose-dependent relationship between plasma homocysteine levels and the number of
cigarettes smoked per day. This relationship was found in all ages and gender groups and
was still strong after adjusting for several potential confounders.155 This dose-dependent
association has been demonstrated in several other large cohorts. 46, 73, 93
A suggested mechanism behind the plasma homocysteine/cigarette-cotinine
relationship is that the free radicals in smoke provoke oxidative stress which affects the
redox status of homocysteine. 194
The reversibility of increased plasma homocysteine with cigarette smoke is
uncertain. There are some studies that found cessation of smoking decreases plasma
homocysteine 178, while others have not.188 This disagreement might suggest that the
relationship between plasma homocysteine and smoking is not (or is only partially)
dependent on the actual smoke itself, but rather, is due to other behavioral traits in
smokers.155 Smokers tend to have a diet containing less vegetables, more fat 150, and less
vitamins 146 when compared to non-smokers.
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Coffee Consumption
Of all the life-style determinants measured in the Hordaland Homocysteine Study,
heavy coffee consumption is among one of the strongest. 136 It appears that this
relationship is only present in heavy coffee consumers. In the ARIC study, no
relationship was found between plasma homocysteine levels and moderate coffee
consumption. Only after four or more cups a day was plasma homocysteine elevation
present. 182 The Hordaland study found that more than six cups a day will provoke a
plasma homocysteine level 2-3μM/L higher than coffee abstainers. Decaffeinated coffee
did not raise plasma homocysteine levels, which suggests that caffeine might be the
culprit in this relationship. 136 Caffeine’s effect on plasma homocysteine might be related
to its influence on the cardiovascular system or kidney function.88, 198 With that said, it is
known that heavy coffee consumption is typically related to an unhealthy life-style and
poor nutritional status. 91 When examining three different life-style determinants together,
it was found that smokers who consumed a high amount of coffee and a low amount of
folic acid had anywhere between 3.2-4.8μM/L higher plasma homocysteine than
individuals with a contrasting lifestyle. 137
Alcohol Consumption
Alcohol seems to have a similar effect as coffee on plasma homocysteine levels.
In the NHANES III study, mild drinkers (one to thirty drinks per month) had plasma
homocysteine levels that resembled that of alcohol abstainers. At higher levels, alcohol
consumption was positively correlated with plasma homocysteine levels. There was also
- 24 -
a correlation between the type of alcohol consumed and plasma homocysteine levels.
Both beer and wine were not correlated to plasma homocysteine, while hard alcohol
was.68 In another study, alcohol consumption forms a U-shaped curve with plasma
homocysteine reduction up to fourteen drinks per week. Above that level, plasma
homocysteine levels began to rise. 155 In a recent study, it was shown that alcohol
consumption could have detrimental effects on both folic acid and B-12 status. 72 This
suggests that alcohol consumption might raise plasma homocysteine levels by hindering
the remethylation pathway in homocysteine metabolism.
Exercise
While the relationship is obscure and still debatable, there have been several
studies showing a weak relationship between exercise and plasma homocysteine levels.
This relationship was the most pronounced in the elderly, showing a 1 μM/L difference
between sedentary and exercising subjects. Exercise was shown to reduce some of the
skewness in the distribution curve, which might suggest that it affects mainly those with
hyperhomocysteinemia. 139 The inverse relationship between physical activity and plasma
homocysteine is not seen in all studies. 46 90 In the Hordaland study, an influence of BMI
was observed as the subjects with a low BMI had an inverse relationship between plasma
homocysteine and exercise, while those with a very high BMI had a positive relationship.
139 It has not been shown if exercise directly influences plasma homocysteine or if the
association reflects healthy lifestyle differences. 155 The relationship between plasma
homocysteine and exercise is unclear and more research needs to be conducted to
confirm any results.
- 25 -
Obesity
As stated previously, the relationship between obesity and plasma homocysteine
is currently obscure. There have not been many studies examining this topic; however,
those studies that have been completed have come to different conclusions. Although
obesity is a well-known risk factor for cardiovascular disease, it is uncertain if obesity
also affects the homocysteine metabolic pathway. Currently, the findings are equivocal
for this.
In a study comparing obesity, hypertension, and plasma homocysteine levels, it
was found that obesity had a positive correlation with plasma homocysteine. The average
plasma homocysteine level in the non-obese normotensives was 8.81 μM/L. The obese
normotensives had an average level of 10.71 μM/L, which was significantly higher (p
< .001) than the non-obese. Within the hypertensives, the same trend was observed. The
non-obese had an average of 8.85μM/L while the obese had an average of 14.85μM/L,
which was also significant (p <.001).99
The same research group did a similar study in diabetic patients with similar
results. The healthy control group had an average plasma homocysteine level of 8.5μM/L.
The non-obese diabetic patients had an average of 10.4μM/L, while the obese diabetics
had an average of 13.2μM/L, both of which were significantly higher than the control
group (p <.001). This suggests that obesity, as well as diabetes, might be causes of
elevated plasma homocysteine levels.100
In a study of cardiovascular disease patients, it was found that both BMI and
waist-to-hip ratio (WHR) were significantly correlated with plasma homocysteine
- 26 -
levels.37 Another study of cardiovascular disease (coronary heart disease in this instance)
patients confirmed that WHR was significantly correlated to plasma homocysteine, but
failed to do so with BMI. The researchers suggested that a possible explanation would be
the detrimental effects that central obesity has on the cardiovascular system. 107
In a German study of 500 healthy participants, it was found that plasma
homocysteine concentrations of the total study group correlated positively with lean body
mass (LBM) (p < .0001) but not fat mass, or BMI. 153 These results were confirmed
findings from another large European Cohort that found a positive correlation between
LBM and plasma homocysteine levels, but not with BMI. 51 In a third study that had very
similar findings, the authors suggested that LBM might be the main variable that affects
the male-female differences in plasma homocysteine levels.18 It is suggested that the
relationship between LBM and homocysteine is attributable to an increased protein mass
relative to body size which could increase circulating methionine and homocysteine.
Creatine synthesis is one of the most important reactions that require methyl groups from
methionine. The greater amount of methionine that donates its methyl groups, the more
homocysteine will be produced.18
It appears that most of the studies done on healthy volunteers show no correlation
between BMI, fat-mass, or obesity and plasma homocysteine, while studies done on
diseased populations do display this connection. It might be the case that some of the
conditions mentioned are confounders in the plasma homocysteine/obesity relationship.
However, it is clear that the relationship between obesity and plasma homocysteine needs
to be investigated further.
- 27 -
HOMOCYSTEINE AND CARDIOVASCULAR DISEASE
Though there is no known biological role of homocysteine, elevated levels of this
amino acid have been shown to be an emerging risk factor for cardiovascular disease.56, 77
This topic is debatable in the literature today, as some incredibly compelling studies have
shown that plasma homocysteine is an independent risk factor for cardiovascular disease.
On the other hand, further studies have found that the relationship is more casual and that
plasma homocysteine is merely a marker of cardiovascular disease.
Homocystinuria and Cardiovascular Disease
As discussed previously, homocystinuria is a severe elevation of plasma
homocysteine levels. In the late 1960s, reports of homocystinuria in children with
thromboembolisms and arteriosclerosis led researchers to believe that plasma
homocysteine is an instigator of vascular and thrombotic disease.119 Though it is still
debatable if moderately elevated plasma homocysteine levels are an independent risk
factor for cardiovascular disease, it is very well agreed upon that homocystinuria levels
are a risk factor. Even the researchers that seem to side with the argument that mild
hyperhomocysteinemia is just a marker of cardiovascular disease, agree that
homocystinuria plays a direct role in vascular and thrombotic disease.124 One researcher
went as far as to say, “This condition is unquestionably associated with precocious
atherosclerosis and extensive arterial thrombosis.”40 The most common causes of death
and morbidity in patients with homocystinuria is thromboembolisms, followed by strokes,
peripheral arterial thrombosis, and heart attacks.40 The findings of patients with
- 28 -
homocystinuria are different than patients with hyperlipidemia. Homocystinuria patients
will have a loosening of the internal elastic lamina, intimal hyperplasia, and narrowing of
the arterial lumen.120, 138, 196 Furthermore, patients with homocystinuria have been shown
to develop drastic arterial intimal thickening and fibrous plaques.104, 119, 158 The
proliferation of these fibrous plaques is caused by hyperplasia of smooth muscle cells and
deposition of collagen fibers. Deposition of cholesterol and lipids in these patients is only
occasionally observed.121 It has been shown that if left untreated, 50% of homocystinuria
patients will experience a thromboembolic event and 20% will die before the age of
thirty.130, 138 On the other hand, in a study it was shown that if homocystinuria treatment
can reduce plasma levels below 20 μM/L, vascular events can be drastically reduced.
Throughout the course of the study214, only two vascular events occurred after lowering
plasma homocysteine levels. Surprisingly, it was estimated that without the plasma
homocysteine lowering treatment, twenty-one events would have occurred.214 Rapid
onset of severe cardiovascular disease has been shown after injecting large quantities of
homocysteine into laboratory animals.81 This experiment suggests that homocysteine
itself might have a damaging effect on blood vessels.186
Hyperhomocysteinemia and Cardiovascular Disease
Since the original literature on this topic came out in the 1960s, a great deal of
attention has been focused on determining the relationship between plasma homocysteine
and cardiovascular disease. Hundreds of studies have followed, most of which have been
done in the last fifteen years.138 While these studies have shown an undeniable
relationship between the two factors, the exact nature of that relationship is still uncertain.
- 29 -
The common belief, after the 1960s studies, was that plasma homocysteine was an
independent risk factor for cardiovascular disease. This belief was coined the
“homocysteine hypothesis of arterioscerosis.”40
A majority of early studies strongly support the homocysteine hypothesis. These
studies found that plasma homocysteine was a strong and independent risk factor for
cardiovascular disease.26, 80, 158, 195 Boushey et al conducted a classic meta-analysis that
included three prospective and six population-based case-control studies that were
considered high quality. An additional five cross-sectional and thirteen other case-control
studies were also incorporated.26 This analysis found that for a 5 μM/L increment in
plasma homocysteine, the odds ratio for CAD was 1.6 in men and 1.8 in women. This
risk elevation is similar to what is seen with a 20 mg/dL increase in total cholesterol. The
authors predicted that roughly 10% of the populations risk for CAD can be attributed to
high plasma homocysteine. In addition, the odds ratio for cerebrovascular disease at a 5
μM/L plasma homocysteine elevation level was 1.5.26 This study investigated the effect
of increased folic acid consumption and found that approximately 200 micrograms/day
reduced plasma homocysteine levels by 4 μM/L, while it also predicted that 13,500-
50,000 CAD deaths could be avoided annually with widespread folic acid
supplementation.26 A more recent study found that a high dosage of folic acid (5-10
mg/day) will reverse endothelial dysfunction in CAD patients.125 The Boushey et al
analysis found that plasma homocysteine should be considered an independent graded
risk factor for cardiovascular disease.26
A more recent meta-analysis found that people with lower than normal plasma
homocysteine levels have a 11% lower heart disease risk and a 19% lower stroke risk
- 30 -
after adjusted for all potential confounding variables.3 A separate finding estimated that a
reduction of plasma homocysteine by 3 μM/L would reduce the risk of cardiovascular
disease by as much as 16%.207
The other school of thought that opposes the homocysteine hypothesis believes
that plasma homocysteine is a mere marker of cardiovascular disease. The main
reasoning behind this belief is that retrospective studies tend to show stronger
associations between plasma homocysteine and cardiovascular disease than prospective
studies. While there have been several prospective studies12, 25, 145, 161, 208 that have shown
a strong correlation between the two factors, most large, well conducted prospective
studies show weaker38, 73, 176, 177, 196 or no association at all between plasma homocysteine
and cardiovascular disease.57, 65 The idea that retrospective studies have more consistently
shown positive associations between plasma homocysteine and cardiovascular disease,
while prospective studies have been less consistent might point to the conclusion that
plasma homocysteine is merely an indicator, as opposed to an instigator of cardiovascular
disease.40, 101, 124
While there is still some debate about what comes first, high plasma
homocysteine or cardiovascular disease, it is widely accepted that once a patient has
cardiovascular disease, plasma homocysteine plays a very large role in mortality and
reoccurrences of vascular complications. A study conducted by Nygard et al followed
600 CAD patients over the course of five years. The patients that had the highest baseline
HCY level had less than a 70% survival rate while over 90% of those with the lowest
baseline plasma homocysteine levels were still alive. It was determined in these patients
- 31 -
with cardiovascular disease that plasma homocysteine was the strongest modifiable
determinant of mortality.138
Mechanisms
While there seems to be strong evidence supporting and opposing the
“homocysteine hypothesis of arteriosclerosis,” it is well agreed upon that there is a
relationship between plasma homocysteine and cardiovascular disease. In order to
investigate this relationship, it is important to examine the biological plausibility, as well
as the mechanisms in which plasma homocysteine could possibly relate to cardiovascular
disease.
Cytotoxicity
Extremely high levels of plasma homocysteine have been associated with direct
toxicity of endothelial cells in several in vivo and in vitro studies.138 These studies are
supported by a finding of an increase in circulating endothelial cells in cardiovascular
disease that were given a methionine load.86
Inflammatory Response
It is well established that atherosclerosis is at least in part due to chronic
inflammation.104, 138 There have been many published findings that show that plasma
homocysteine enhances the production of several inflammatory cytokines.104 Human
monocytes express a number of different pro-inflammatory cytokines.183 In the
Physicians’ Health Study, plasma homocysteine was significantly correlated to the
concentration of intercellular adhesion molecule ICAM-1.160 In other findings, when
- 32 -
human monocytes were treated for three hours with various concentrations of plasma
homocysteine, it was shown that Tumor necrosis factor-alpha, Interleukin 1 beta,
Interleukin-6, Interleukin-8, and Interleukin-12 expressions were significantly enhanced
1.2-2.0 fold.183 Monocyte chemoattractant protein 1 (MCP-1) is known to enhance
binding and recruitment of monocytes to the sub-endothelial cell space. MCP-1 has also
been shown to increase in cultured human endothelial and smooth muscle cells with an
increased homocysteine level.209
Oxidative Stress and Endothelial Function
Homocysteine contains a thiol group that is readily oxidized to create several
types of reactive oxygen species (ROS).15, 104 It has been proposed that the ROS will react
with endothelial nitric oxide (NO) (a potent vasodilator) and decrease its’ availability.
With a smaller amount of NO available, the endothelium dependent vasodilatation will be
impaired.15, 104, 138 Endothelium function is considered to be a sensitive marker of
vascular pathology and has been shown to be impaired in cardiovascular disease
patients.125 Endothelium function is easily measured using a technique called Flow
Mediated Dilation (FMD). This technique provokes intentional ischemia using a blood
pressure cuff of some sort on the arm to provoke a hyperemic condition downstream. The
pressure is then released and ultrasound technology is used to measure vessel diameter
before and after the increased flow.125 When FMD was measured in children with
homocystinuria, it was found to be significantly impaired (2.8% response) when
compared with controls (9.0% response).36 Another recent study confirmed that CBS
deficient patients with severe hyperhomocysteinemia had impaired NO-mediated FMD,
- 33 -
compared to matched controls.125 This relationship is seen even in more moderate forms
of hyperhomocysteinemia. Fourteen age and sex matched patients with an average of
34.8 μM/L plasma homocysteine had impaired (6.5% response) vasodilatation compared
to controls (10.8% response).219
Smooth Muscle and Collagen Proliferation
As described previously, hyperhomocysteinemia and homocystinuria patients have been
shown to exhibit fibrous plaques in atherosclerosis, opposed to the traditional cholesterol
and lipids seen in the lesions of cardiovascular disease patients with normal plasma
homocysteine levels. A good deal of this plaque is created by the growth of smooth
muscle cells and collagen in the vessel wall.104, 138 It was shown in an in-vitro study that
when exposed to homocysteine, the proliferation of smooth muscle cells was double that
of in non-exposed cells.185
TREATMENTS FOR OBESITY
As the obesity epidemic is rapidly growing, it is imperative to examine methods
to combat and treat obesity. There are numerous different mass loss programs in America
today. Although they vary, programs or techniques can be lumped into one of six
different categories.
1) Diet therapy aims to provoke mass loss by restricting calories consumed on a
daily basis. Low calorie diets (LCD) will typically aim at reducing calories to
1000-1500 per day.1 Very low calorie diets (VLCD) restrict calories even further
to 400-800 per day. 205
- 34 -
2) Exercise therapy aims to reduce mass by increasing the amount of energy
expended. 1, 2
3) Behavioral therapies include strategies that aim to reinforce diet and exercise
therapies. Some of these therapies are cognitive restructuring, self-monitoring,
stimulus control, stress management, and social support.1, 2
4) Combined therapy is using more than one of these strategies simultaneously. This
has been shown to be one of the more effective ways to lose mass.1, 2 In a review
of fifteen RCT’s, there was strong evidence that combination therapy produces
greater mass loss than diet or physical activity alone.1
5) Pharmacotherapy uses FDA approved drugs as an adjunct to any of the above
therapies. Typically, doctors will not prescribe pharmacotherapy unless combined
therapy has been unsuccessfully implemented for at least six months. A BMI of
above 30 kg/m2 is general criteria to receive pharmacotherapy, but doctors can
prescribe it at lower BMI levels with one or more of the following: hypertension,
dyslipidemia, cardiovascular disease, type II diabetes, or sleep apnea.1, 2
6) Mass Loss surgery is often considered the last resort therapy for severe obesity.
Surgery is generally reserved for patients who have unsuccessfully tried the above
therapies and have a BMI of over 40 kg/m2. Patients with a BMI of above 35
kg/m2 sometimes qualify for surgery if co-morbid conditions are present. Most
authorities agree on these standards and will only prescribe surgery if the patient
is suffering from the complications of obesity. 1
- 35 -
Though combined therapies are effective at decreasing mass acutely in
overweight and moderately obese subjects, very rarely does it lead to prolonged and
maintained mass loss. RCT’s suggest that the mass lost will usually be regained unless
some sort of maintenance program is continued indefinitely. 1 Several studies have
indicated that combined therapy, in the severely obese, has limited long term
effectiveness for the vast majority (>90%) of those who attempt to lose mass.205, 212 The
National Institute of Health (NIH) Consensuses Panel claims, “Although a very-low-
calorie diet used under close medical supervision is often effective in the short-term
treatment of clinically severe obesity, these diets alone generally have not been
successful in achieving permanent mass loss.”4
In the Diabetes Prevention Program, 1079 obese (BMI = 33.9 kg/m2) subjects
were put into a rigorous diet and exercise program. Despite the intensive individualized
guidance, after 2.8 years, the patients had only lost 5.6 kilograms. 98
In the Swedish Obese Subjects (SOS) study, 627 morbidly obese patients (BMI =
40.5 kg/m2) underwent conventional treatment (from intensive lifestyle advice to diet and
exercise) and were followed for ten years. Though there was a modest initial mass loss, at
the end of the follow up period, the group had a mass gain of 1.6%.173 Though the study
could be considered a success in that the patients prevented much mass gain, it
established that conventional treatments are typically not able to provide maintained mass
loss in the morbidly obese.
In a review article examining the effects of very low calorie diets, a mass
reduction of 15-25% was typically observed after three to six months. However, after one
year, the average was only 9% mass loss, and after four years it was only 5%. 190 These
- 36 -
results were very comparable to another study that had an average mass loss of 3.0 kg
over four years in 1637 patients treated with a placebo and lifestyle therapy.189
Even pharmacotherapy, which is considered the second line of therapy, has shown
to only provide moderate long-term mass reduction in the morbidly obese. In the Swedish
Xendos trial mentioned above, the group treated with the actual drug had a mass loss of
only 5.8 kg over the 4 years.189 The NIH Consensuses Panel released the following
statement about pharmacotherapy, “Experience with drug therapy for clinically severe
obesity has been disappointing. Although pharmacologic studies of anorexigenic drugs
suggest a short-term benefit, prolonged and sustained mass loss has not been proven with
these agents.” 4
Marielle Bult summarized all of this information well with the statement, “The
long-term effects of diet, exercise, and medical therapy on mass are relatively poor.”31
Mass Reduction Surgery
While it has been shown that most of the “traditional” mass loss methods are
relatively ineffective at treating morbid obesity and maintaining the mass loss, mass
reduction surgery has shown promising results. While the obesity epidemic worsens,
more and more patients have opted for this option of treatment. Between 1993 and 2003,
the number of procedures performed annually rose from under 20,000 to well above
100,000.180
There are several different types of mass reduction surgery. The most common
are: gastric banding, gastric bypass, gastroplasty, and biliopancreatic diverson. 30 All
bariatric surgeries are classified as either restrictive, malabsorptive, or both. Restrictive
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surgeries limit the amount of food a patient can consume by shrinking the size of the
stomach. Malabsorptive surgeries bypass portions of the small intestines, which is where
nutrient absorption occurs. Combination surgeries combine both malabsorptive and
restrictive properties.48 Typically, mass reduction will be greater in malabsorptive and
combination surgeries than in restrictive alone.30 A study done in 2004 estimated that
over 90% of all procedures performed that year were Roux-en-Y gastric bypasses133,
which is considered mainly restrictive, but partially malabsorptive as well.48
Depending on the institution and procedure performed, these surgeries generally
cost anywhere from $20,000 to $50,000, which includes the surgery itself plus any future
costs that could be attributed to the surgery. Additionally, the cost of the surgery may or
may not be covered by insurance. 180
While the varying procedures have different results in regards to mass reduction,
it has been estimated that mass reduction surgeries as a whole will produce a twenty to
fifty kilogram loss. As mentioned before, this is compared to a small mass gain seen in
medically treated control patients.30, 112 44
As described previously, the main criterion for being a surgery candidate is
having a BMI over 40 kg/m2, or 35 kg/m2 with a high-risk condition. Depending on the
surgical center, there are some additional criteria that are often imposed. Some of these
criteria are: an absence of medical or psychological contraindications, strong motivation
to comply with post-surgery regimens, and a good understanding of the risks involved
with the surgery.48 There are several psychiatric disorders that are relatively common in
the morbidly obese and have been shown that, when present, less mass loss than normal
can be expected. Some of these conditions are Axis I or Axis II disorder, disturbed eating
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habits (binge eating), substance abuse, low socioeconomic status, limited social support,
and unrealistic expectations of surgery. 48, 200
ROUX-EN-Y GASTRIC BYPASS SURGERY
As mentioned in the previous section, the roux-en-Y gastric bypass is by far the
most popular of the mass loss surgeries. The surgery is almost always done
laparoscopically. Though the surgery was initially performed by opening the abdominal
cavity, the minimally invasive laparoscopic procedure has been shown to greatly reduce
complications after surgery. 85, 143, 168 In a study examining the outcomes of open
RNYGB compared to laparoscopic RNYGB surgeries preformed in 2005 and 2006, it
was found that practically every category of negative occurrences was statistically less in
the laparoscopic group. The open group had .79% death rate within the first thirty days of
surgery while the laparoscopic group had .17%. “Major” occurrences such as deep wound
infections, organ space infections, pneumonia, renal failure, and many more were over
twice as common at 7.4% in the open group compared to 3.4% in the LRNYGB group.103
Surgery Description
The actual procedure involves creating a small gastric pouch (typically around 15-
50mL) and separating it from the rest of the stomach to severely restrict the amount of
food the patient can eat. The pouch is then anastomosed (joined) with the proximal
jejunum. This segment will form the “roux” limb. The other side (distal) of this limb,
along with the duodenum, will be anastomosed with the distal jejunum. This will lead to
a bypass of a portion of the jejunum, and thus provoking malabsorption in the small
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intestines.31, 85, 115, 168 For a complete description of the surgical procedures, see Higa,
2001.85
Mass Loss
Though surgery results, in regards to mass lost, vary from study to study, it is
expected that patients who undergo RNYGB will lose around 60-70% of their excess
mass in one to two years.64, 168 Excess mass is the actual mass prior to surgery minus the
“ideal mass” for that individual. If the patient does not lose at least 50% of their excess
mass, or keep it off for the course of the study, it is deemed a “mass loss failure.”64 Other
studies suggest that the surgery typically provokes somewhere between a 30% and 40%
mass loss of total body mass in the first year.168
It has been shown that it is very common for the patient to gain some of this mass
back over the course of time. Mass loss is usually maximal after one to two years after
which mass will gradually increase until eight to ten years. Typically after ten years, body
mass will stabilize.31, 168, 174 There can be many causes of mass regain in these patients.
The most common causes are an initial large pouch (.30cc), and initial large stoma
(.14mm), a dilated pouch, dilated stoma, staple line disruption, and increased energy
consumption. On average, roughly 15% of patients will regain enough mass to have
“mass loss failure.”64
Despite the relatively small percentage of patients that regain a large amount of
mass, the majority of patients maintain considerable mass loss for at least ten years (very
few studies follow patients beyond this time frame). In a recent meta-analysis, it was
found that nearly all studies show at least an average of 50-60% excess mass lost at the
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end of the follow-up period. One of the studies had a follow up of sixteen years, two had
a follow up of fourteen years, one had a follow up of ten to twelve years, and four had a
follow up between four and seven years.168 With such long term follow up periods, it
seems clear that for the majority of patients, the RNYGB surgery is a very effective way
to significantly reduce body mass and maintain that mass loss for an extended period of
time.
Side Effects
Despite the significant mass loss that occurs with this surgery, one has to
remember that it is still surgery and has its risks. It is very well known that any type of
gastric surgery has many complications associated with it. Some of these complications
are common and relatively mild, while others are rare, but very serious. Most of the time,
the benefits of the surgery in mass reduction and improved health (to be discussed later)
outweigh these risk for the patient.
The following have been reported as rare complications that occurred in less than
one percent of 1040 patients treated in one surgical center: staple line failure, stenosis at
mesocolon, bleeding requiring transfusion, death, incomplete division of the stomach,
pulmonary embolism, trocar hernia, deep venous thrombosis, pneumonia, and wound
infections. The following were reported in one through five percent of patients in the
same study: stenosis at gastrojejunostomy, internal hernias, gallstones, and marginal
ulcers. The total complication rate for this study was just under fifteen percent.84 A
different study examining 4,631 LRNYGB patients found similar results with most
complications occurring under one percent of the time. 103 With that said, when looking at
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all complications grouped together, that study found that roughly seven percent of
patients will experience some sort of complication in the first thirty days. Close to four
percent of patients will have to return to the operating room due to one of the
complications.103
However, another study examining 235 RNYGB patients found a few more
complications. Over twenty three percent of the patients in that particular study reported
having some sort of complication.143 There could be a couple of reasons for this
discrepancy. The first possibility could be is how the studies defined a complication, as
well as how long the patients were followed. The study that had only a seven percent
complication rate was only found in the first thirty days. This study also did not seem to
report the less severe complications such as nausea, vomiting, dehydration, or the
dumping syndrome.103 On the other hand, the study that had a twenty three percent
complication rate had an average follow up of over twelve months and included
complications such as prolonged postoperative stay, dehydration, as well as
nausea/vomiting. When this study separated their “early complications” from their “late
complications,” they found just above nine percent occurred in the “early” phase143,
which is much closer to that which was found in the first study.103
Some of these “less serious” complications of surgery seem to be rather common.
The dumping syndrome, which is a group of symptoms that include facial flushing,
lightheadedness, palpitations, fatigue, and diarrhea can occur in up to 70% of patients.
This syndrome is usually provoked by consuming foods high in sugar.181 Up to 50% of
patients can experience nausea and vomiting. These symptoms are usually the result of
eating too much, too quickly.122
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Vitamin Deficiencies
The whole concept behind the malabsorptive properties of the RNYGB surgery is
to not allow enough time for the nutrients to fully absorb in the digestive tract, and
therefore provoke mass loss by providing less energy for the body to use. This is a good
thing when it comes to mass loss, but along with the malabsorption of energy containing
nutrients, the other nutrients essential for life functions also do not get absorbed at their
normal rate. This malabsorption often leads to a deficiency in particular nutrients. More
importantly, this situation is aggravated by the restrictive properties of the surgery, which
decreases the total amount of nutrients that could potentially be absorbed.43 The most
common nutrient deficiencies in RNYGB surgery are vitamin B-12, iron, and thiamine.
115 A less common, but still possible, deficiency resulting from surgery is folic acid.115
Vitamin B-12
Vitamin B-12 (cobalamin) deficiency has been defined as a plasma level under
150pg/ml-200pg/ml.168, 202 Cobalamin deficiency is rather rare in the general population
because it is widely available in animal products. Additionally, it is stored in large
quantities in the liver.159 That being said, it is quite common in RNYGB surgery patients
to have this vitamin shortage. While cobalamin deficiency has been seen in as many as
64% of surgery patients 78, it is more commonly observed in 20-30% of patients,
depending on the limit definition used, as well as the presence of any type of post surgery
supplementation.168 Some possible factors that lead to B-12 deficiencies include
achlorhydria, which prevents B-12 from being separated from food, and poor production
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or secretion of intrinsic factor, which is needed for the nutrient to be absorbed.43, 159, 168
Post-surgery, some patients become intolerant of meat and dairy, which are key sources
of B-12 and will contribute to its deficiency as well.115, 168 Another major factor resulting
in B-12 deficiency is that other chemicals that release cobalamin from food, such as
hydrochloric acid and pepsin that reside in the stomach, are rarely found in the small
gastric pouch created during surgery. This makes the absorption of protein bound
cobalamin difficult.159 Absorption of crystalline B-12 does not seem to be affected by the
surgery159, which is why sufficient supplementation will usually restore cobalamin levels.
Supplementation has been shown to be affective at preventing cobalamin
deficiencies in RNYGB patients. A study found that roughly 350 μg of crystalline
cobalamin was necessary to prevent deficiency post-surgery.159 Most studies examined
recommend 500-600 μg to sufficiently cover this amount. 29, 159, 168 It was found that this
recommended amount will raise over 80% of deficient patients into the normal range.29 If
the patient does not respond to oral supplementation, 1000-2000 μg administered
intramuscularly once every month will correct the majority of the remaining 20%.115, 168,
202
Folic Acid
While not as common as B-12 deficiency, folic acid deficiency is equally
important in the metabolism of homocysteine and needs to be addressed if present in
post-surgical patients. Folate is a generic term for a water-soluble B-complex vitamin. It
is essential for DNA synthesis, erythrocyte formation, and a cofactor in many other
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metabolic pathways apart from homocysteine. Maintaining sufficient levels of folic acid
is important because deficiency can also lead to megaloblastic anemia.115
Folic acid is preferentially absorbed in the proximal portion of the small
intestine.168 For this reason, folic acid status might be strained after surgery because this
is the part of the intestine that gets bypassed. However, it has been shown that the small
intestines are able to adapt following surgery and are able to absorb folic acid along the
entire small intestine.7
Folic acid deficiency is normally defined by any level under 3 ng/mL.202 The
normal range for plasma folic acid levels is 6-25ng/mL.115 Deficiency is seen in 9-35% of
surgeries.28 In a recent study, the mean folic acid level of all patients continually rose for
each follow-up visit. After three years the mean folic acid level was 12.2 ng/mL
compared to 10.8 ng/mL pre-operation.202 While it appears that folic acid deficiency is
typically not a big concern, it is still important to be monitored post-surgery and treated if
present. Brolin et al found that a 400 μg supplement of folic acid is adequate to correct
almost any deficiency.29
Surgical Experience
Another large factor to examine when looking at post-surgery complications is the
surgeon’s experience. In a ground-breaking study, Flum and colleagues found that early
mortality after surgery was directly linked to surgical inexperience with the procedure.
Patients that had inexperienced surgeons were nearly five times more likely to die within
thirty days of surgery, compared to patients of experienced surgeons. Quantitatively,
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surgeons that had less than fifty surgeries had a death rate around six percent compared to
less than half a percent for those with 100 or more surgeries in experience.63
Health Benefits
Other than mass loss, gastric bypass surgery has typically been shown to
drastically improve the patients overall health.
Mortality
In one of the very few studies that examine long-term mortality rates after gastric
bypass surgery, Adams et al analyzed data from 9949 surgical patients that received
surgery from 1984-2002 in a Utah surgical practice, as well as 9628 matched controls.
The matching was based off of sex, BMI, age, and year. Surprisingly, between the two
groups, the researchers found the same mortality rate in the first year after surgery
between the two groups (0.53% in surgery group compared to 0.52% in the control
group).5 This can be expected from the death rates discussed previously in the
complication section above. This study brings to light the idea that the risk of dying from
complications post-surgery is very close to the risk of not having the surgery at all.
The Adams et al study proceeded to follow the patients for an average 7.1 years
(>70,000 person years). After the follow up period, 213 surgical patients had died
compared to 321 from the control group. This is the equivalent of 37.6 and 57.1
respectively per 10,000 person years. When the cause of death was accounted for, 150
surgical patients died from some sort of disease compared to 285 controls (26.5 and 50.7
per 10,000 person years). Cardiovascular disease claimed fifty-five deaths in the surgery
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group compared to 104 controls. Only two surgical patients died from diabetes, while
nineteen controls died from the same disease. More than twice as many people (73
compared to 31) died from cancer in the control group. The only surprising statistic was
that there were twice as many deaths (sixty-three compared to thirty-six) caused by non-
disease reasons in the surgery group. This figure is greatly influenced by suicide rate,
which was three times greater in the surgical group. The authors suggest that a possible
explanation for this is that a majority of obese persons have unrecognizable mental health
problems that may come to surface after surgery. They recommend that further studies
explore this topic more in-depth and provide more rigid pre and post surgical
psychological counseling to patients.5
In a similar study, another group of researchers found similar results in regards to
overall mortality rates. There was a five-year follow-up period of roughly 1000 surgical
patients and 6000 controls. Overall mortality rate for surgical patients was .68%
compared with 6.17% for the controls. The most notable risk reductions were in
cardiovascular disease (including hypertension), endocrinologic conditions (including
diabetes), and respiratory conditions.39
In the same study that found that experience of the surgeon relates directly to
thirty day mortality, it was found that ten year survival of bariatric patients was relatively
high at 91.2%. At fifteen years follow-up, 11.8% of the surgical patients had died,
compared to 16.3% of non-operated patients.63
It appears relatively clear that roux-en-Y gastric bypass surgery is very effective
at prolonging life in the morbidly obese. A reason for this extension could be the
reduction in many chronic disease risk factors. As discussed previously, it is well proven
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that obesity drastically increases many of these risk factors, so it would seem probable
that reduction of obesity would, in turn, lead to the reduction of risk factors as well.
Diabetes
Of all the benefits that come with RNYGB surgery, the improvement, or complete
resolution of diabetes, could be one of the most pronounced. In a large meta-analysis of
several studies, Shah et al.168 found that the majority of all diabetic patients having
surgery would see their condition completely resolved. One of the studies examined
found that 32% of the surgery patients were taking medication for diabetes prior to
surgery, while only 9% were still taking at the last observed follow-up.110 Pories et al
found that 83% of patients had recovery of diabetes after adequate follow-up.149 In a
study of 1,025 patients, Sugerman et al found that 86% of diabetics no longer had
diabetes after a seven year follow-up.184 In the SOS study after two years, 72% of
diabetics had complete resolution.173 In a compilation of these studies, as well as others,
it was found that the average was 83.8% of diabetics saw complete resolution, while
90.6% saw resolution or improvement.30 It is very clear that for morbidly obese diabetic
patients, not only will the surgery benefit their mass, but chances are pretty high that it
will resolve their diabetes.
Hyperlipidemia
While the diabetes data is very promising and dramatic, improvement in lipid
levels after surgery is comparable, if not more promising. In the same compilation of
studies, it was found that 94% of patients saw an improvement in hyperlipidemia, 95%
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improvement in hypercholesterolemia, and 94.1% in hypertriglyceridemia.30 In addition,
the SOS study found that those with low HDL cholesterol saw that condition improve to
normal ranges in 73% of patients.173
Hypertension
While the results are not as pronounced for hypertension, there still has been very
well documented improvements with this condition after surgery as well. The Sugarman
study found that 69%, 66%, and 51% of hypertensives saw resolution of their condition
after 1, 5-7, and 10-12 years respectively.184 The Buchwald et al meta-analysis found that
75.4% had resolution, while 87.1% saw improvement or resolution.30 For some reason
the SOS study had only 34% and 19% resolution at two and ten years post-surgery,
respectively. However, when that result is compared to the 21% and 11% resolution rates
of medically/lifestyle treated controls, the surgery still provides a benefit to the
surgery.173
Sleep Apnea
Obstructive sleep apnea is quite common in the morbidly obese. As many as 77%
of all patients seeking bariatric surgery have sleep apnea.140, 201 This condition is often
overlooked in the morbidly obese, but it has been shown that sleep apnea is linked to
intermittent hypoxia, ventricular dysfunction, increased hospital stay, a risk factor for
heart disease, as well as a risk for pulmonary complications after surgery.16, 172, 220 The
Buchwald meta-analysis found 86.6% resolution and 94.9% improved or resolved.30
Another study found that even with a small amount of mass lost at one month post-
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surgery significantly improved symptoms of sleep apnea. After nine months, none of the
patients previously using CPAP (a machine used to treat sleep apnea) still required that
therapy.140
Homocysteine
It is well understood that RNYGB surgery greatly improves several well-known
factors for cardiovascular disease. With plasma homocysteine being an emerging, and
potentially independent, risk factor for cardiovascular disease, it is important to study the
effect that this surgery has on plasma homocysteine. Very few studies have examined
plasma homocysteine levels after bypass surgery and those that have examined have been
very inconclusive.
In a poorly designed retrospective study looking at all cardiovascular disease risk
factors in 73 women after gastric banding surgery, researchers were surprised that plasma
homocysteine levels were higher in the surgery compared to the control group (13.3
μM/L compared to 9.2 μM/L (p<.001)). There was no pre-surgical measurements for the
index group and did not have any measure of the factors essential to plasma
homocysteine metabolism such as B-12 and folic acid status.94 In addition, the study was
examining gastric banding which typically does not provoke as drastic of reduction in
mass as RYNGB.
A better-designed, older study followed fifty three gastroplasty patients for one
year following their surgery. The patients were predominantly female with an average
BMI of 42.0 (±1.0). The patients were followed for a total of one year and lost an
average of 32 kg (28% loss). This study measured folic acid and B-12 and accounted for
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these vitamins in their analysis. They found that B-12 status did not change over the year,
although information on B-12 supplementation was not described in the article. On the
other hand, folic acid status decreased 20% during the year. Mean plasma homocysteine
levels increased from 9.9 μM/L to 12.8 μM/L (p<.0001). Forty of fifty-three patients saw
their plasma homocysteine increase. The authors assumed that this increase was directly
related to folic acid status.24 The big downfall of this study was the ambiguity of vitamin
supplementation. In order to observe if changes in mass effect plasma homocysteine,
vitamin status must be controlled.
A third study tracked 12 morbidly obese patients for a year after gastric banding.
Mean weight loss was 32kg (26.7%) after twelve months. This study found that even
when cobalamin and folic acid status remain constant, plasma homocysteine levels
increased 10.2 to 12.1 μM/L (p=.040) at one year after gastroplasty. Surprisingly, the
patients did not take any type of vitamin supplementation in this study, but somehow
retained their vitamin status.170
On the contrary, a recent study has shown that a decrease in plasma homocysteine
occurs after gastric bypass surgery. Patients (n=101, predominantly female) receiving
gastric bypass surgery were followed for one year after their surgery. They were given
1000 μg/day of vitamin B-12. After one year, it was found that plasma homocysteine
levels fell from 10.2 μM/L to 8.4 μM/L (p<.0001). Other than the mention of the B-12
supplementation, vitamin status was not addressed.217
With such contradicting findings, it is apparent that the literature is inconclusive
about the impacts of bariatric surgery on plasma homocysteine. Some reasons for these
limitations and discrepancies are that the first three studies were examining different
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types of surgery. Some studies were poorly designed and failed to examine key
components of plasma homocysteine metabolism.
Very little is known about the specific effects RNYGB surgery and its respective
loss in mass has on the cardiovascular disease risk factor plasma homocysteine. With
such a large proportion of the American population being obese and the high prevalence
of cardiovascular disease, it is important to examine if the surgery alters serum
homocysteine levels.
SPECIFIC AIMS OF THE STUDY
The primary aim of this study was to examine the change in serum homocysteine
levels following laparoscopic Roux-en-Y gastric bypass surgery in morbidly obese
patients. A secondary aim was to study the associations between concentrations of
homocysteine at baseline and 12 months following surgery, with known determinants of
its metabolism, including vitamin B-12 and folic acid, as well as anthropometric
measures (body mass and body mass index) at the respective time points. Additionally,
correlations were determined between changes during a one-year follow-up after bypass
surgery in concentrations of homocysteine, vitamin B-12, and folic acid, as well as body
mass, and body mass index .
HYPOTHESES
Based upon the research presented in the literature review, the hypothesis for the
primary aim is that serum homocysteine concentrations will decrease following the
Roux-en-Y surgery. For the secondary aims, serum homocysteine at baseline and 12
- 52 -
months post-surgery will be negatively related to vitamin status with lower homocysteine
associated with higher vitamin levels at the respective time points. Changes in
homocysteine will be negatively related to total mass/BMI loss with decreased
homocysteine associated with greater mass/BMI loss. Changes in homocysteine will also
be negatively correlated to changes in vitamin status. The more vitamin B-12 and folic
acid increase, the more homocysteine will decrease.
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METHODS
The current analyses is an ancillary substudy to an investigation that was
conducted from 2005-2007. The original study was named the Laparoscopic Obesity
Surgery Intensive Treatment (LOSE-IT) study. The primary purpose of LOSE-IT was to
observe the changes of bariatric surgery (Laparoscopic Roux-en-Y gastric bypass) on
physical function measures. Methods for LOSE-IT are described in this section. The
current substudy used serum samples and data collected from the original study to
examine the changes resulting from the surgery on homocysteine, vitamin B-12, and folic
acid.
RECRUITMENT AND ELIGIBILITY REQUIREMENTS
Recruitment for the original study took place at Wake Forest University Baptist
Medical Center through the general surgery office of Dr. Adolfo Fernandez. Patients
were scheduled for surgery prior to learning about and being recruited for the study. Dr.
Fernandez and his staff (Susan Butler, R.N.) assessed the patient’s eligibility at an early
screening visit for the surgery.
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Men and women were eligible for the study if they had a BMI ≥ 40.0 kg/m2 or ≥
35.0 kg/m2 with an obesity related comorbidity, such as hypertension, dyslipidemia, or
diabetes. Since the primary purpose of the original study was to look at the effect of
RNYGB on physical function, an additional inclusion criteria was that the patients have a
self-reported difficulty in performing at least one of the following activities:
lifting/carrying groceries, walking one-quarter of a mile, getting in and out of a chair, and
climbing up or down stairs. They were also to be sedentary with no more than twenty
minutes of exercise on two or fewer days per week. Since computed tomography (CT)
scans were taken in the larger trial to assess regional body composition, an exclusion
criteria was pregnancy at any point of the baseline or follow-up testing.
INFORMED CONSENT
If the surgery patient met the above eligibility requirements, Dr. Fernandez and/or
his staff informed the patient of the study and administered the informed consent. The
Institutional Review Board at Wake Forest University approved the study. Participants
were alerted of potential risks associated with participating in the study. They were
encouraged to contact the project coordinator if they experienced any negative effects of
the testing procedures. The risk of the testing procedures was very minimal. Blood drawn
at each visit might cause slight discomfort, bleeding and bruising, with minimal risk for
infection and fainting.
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STUDY PROCEDURES AND DATA COLLECTION
A total of twenty-eight participants were recruited and consented to the original
study. Clinic staff obtained health history, current medications, and demographic
information for all patients. There were two baseline appointments for each participant.
Each baseline appointment occurred between four and twenty four days prior to receiving
gastric bypass surgery. The average time between baseline measures and surgery was 7.7
days. Testing occurred at the Geriatric Research Center (GRC) located at Wake Forest
University Baptist Medical Center (WFUBMC). A twelve-hour fasting blood sample was
obtained at this time for measures of lipids, hormones, vitamin measurements,
homocysteine, and inflammatory cytokines. This blood sample was stored as serum at -70
degrees Celsius. Participant also reported to the Geriatric General Clinical Research
Center (GGCRC) during this visit. Using standard techniques, the GGCRC staff obtained
baseline measures of blood pressure, body composition, height, and mass. Both height
and mass were obtained with shoes and outer garments removed. Body mass index was
then calculated from these measures. Body composition (fat mass and lean body mass), in
both absolute and relative amounts, was measured using bioelectrical impedance
assessment (BIA) (RJL Quantum II Desktop, Clinton Township, Michigan) with
participants in a supine position. The GGCRC nutritionist also met with the participant to
give instructions on completing dietary records. Four-days of dietary intake were
recorded over a two-week period. Nutrient analyses for these records were determined
using Nutrient Data System (NDS) software.
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Participants underwent laparoscopic Roux-en-Y gastric bypass surgery. Surgery
was performed by Dr. Fernandez using standard Roux-en-Y procedures for all surgeries.
For a complete description of the surgical procedures, see Higa, 2001.85
Follow-up assessments were conducted at three weeks, three months, six months,
and one year post surgery. The follow-up appointments consisted of all measures
obtained at baseline. Food diaries were mailed to the participants prior to each follow-up
visit for completion. The participants returned these completed diaries at their scheduled
appointment.
NUTRITIONAL SUPPLEMENTATION
As part of standard post-surgery care to prevent nutritional insufficiencies,
participants were prescribed a regimen of nutritional supplements. Patients were told to
take a daily multivitamin and mineral supplement, as well as a calcium supplement (600
mg) with vitamin D twice daily, and a daily Vitamin B12 supplement of 500 μg. If the
participant did not respond well to the B-12 supplement, they received a monthly
intramuscular injection of 1000 μg of B-12. Iron supplements were not routinely required,
unless the patient had post-operative anemia. The multi-vitamin was in a chewable form
and was used twice daily for the first month post-surgery, but after one month the patient
converted to a regular multi-vitamin. Supplementation compliance was not monitored.
However, blood concentrations of vitamin B-12 and folate were assessed.
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HOMOCYSTEINE MEASUREMENT
Serum samples from each participant’s five visits were analyzed in the the lab of
Carolina Liquid Chemistries (CLC) (Winston-Salem, NC) which is located in the Wake
Forest University Research Park. The CLC Biolis 24i chemistry analyzer was used to
perform these assays. This analyzer used a recombinant enzymatic cycling assay (RECA).
The analyzer used a reaction of homocysteine and L-serine to form cystathionine
catalyzed by cystathionine synthase. This reaction was followed by the conversion of
cystathionine to homocysteine, pyruvate, and ammonia catalyzed by cystathionine β-
lyase (CBL). The rate of pyruvate production was measured by inclusion of lactate
dehydrogenase and nicotinamide adenine dinucleotide and was directly proportional to
the concentration of homocysteine. (Carolina Liquid Chemistries, HCY test kit literature)
The analytical range for this assay is 0.00-86.6 μM/L. No sample exceeded this
range so no dilutions were necessary. The reported sensitivity for the assay is 0.0 μM/L.
Twenty replicates in two different levels (high and low) were run to confirm precision.
The standard deviation for these levels was .24 and .27 μM/L which had a CV% of 3.3%
and 0.8% respectively.
FOLIC ACID MEASURMENTS
Folic acid assays were preformed in the gerontology laboratory of Dr. Barbara
Nicklas, located in the Nutrition building at Wake Forest University Baptist Medical
Center. This assay was run using the IMMULITE 1000 analyzer. Folic acid was a boil,
competitive, liquid-phase, ligand-labeled, protein-binding chemiluminescent assay. It
used in situ immobilization and contained an anti-ligand detection system. A polystyrene
- 58 -
bead, which was enclosed in the IMMULITE test units, was covered in a murine
monoclonal antibody specific for folic acid binding proteins. (IMMULITE Folic Acid test
kit literature)
The reference range for the assay was 3.00-17.00 ng/mL. The analytical range
was 1.00-24.00 ng/mL. If a sample exceeded these ranges, it was diluted and analyzed
again. The calculated sensitivity of the assay using six replicates was 0.00 ng/mL and the
test claim sensitivity was 0.80 ng/mL. These measurements were well below 1.60 ng/mL,
which was deemed the acceptable limit. Reportable range verification was run at four
different levels with a minimum of three trials per level (six at level one). The %CV for
each level never exceeded 2.52% which fell within acceptable limits. Ten more samples
were run in three additional levels to confirm precision and accuracy. The CV% for each
of those levels was 2.75%, 2.22%, and 2.08%, all of which were within acceptable limits.
VITAMIN B-12 MEASUREMENTS
Vitamin B-12 assays were also preformed in the gerontology lab at WFUBMC
using the IMMULITE 1000 analyzer. Similar to folic acid, the vitamin B-12 assay is a
solid-phase, competitive chemiluminescent enzyme immunoassay. This assay involved a
preliminary heat denaturation step to release the vitamin B-12 from its carrier protein.
The sample was then treated with hog intrinsic factor and introduced to a polystyrene
bead coated with a B12 analog. (IMMULITE B12 test kit literature)
The reference range for the assay was 174.00-878.00 pg/mL. The analytical range
was 150.00-1,000.00 pg/mL. If a sample exceeded these ranges, it was diluted and
analyzed again. The calculated sensitivity of the assay using six replicates was 42.22
- 59 -
pg/mL and the test claim sensitivity was 125.00 pg/mL. 250.00 pg/mL was deemed the
acceptable limit. Reportable range verification was run at four different levels with a
minimum of three trials per level (six at level one). The %CV for each level never
exceeded 6.4% which fell within acceptable limits. Ten more samples were run in three
additional levels to confirm precision and accuracy. The CV% for each of those levels
was 8.13%, 4.91%, and 3.80%, all of which were within acceptable limits.
DROP OUTS
A total of 28 participants were recruited for the original study. One participant
opted out of the surgery after baseline data were collected. Two patients dropped out of
the study due to complications resulting from the surgery. An additional four participants
dropped out as they were no longer being followed by the surgeon or personal reasons
(lack of time and no child care).
These dropouts led to a total of twenty participants with serum collected from all
visits. Since only one of these participants was male, that patient was excluded to only
examine females.
STATISTICAL ANALYSIS
All data were analyzed for normality through descriptive statistics and frequency
histograms. Since serum homocysteine levels and vitamin B-12 were skewed, the data
were transformed using the natural log. While all analyses were done using the log
transformed data for both variables, raw data were reported for ease in understanding.
Scatterplots for demonstrating correlational data are shown with the transformed data.
Descriptive statistics, including means, ranges, and standard deviations were calculated
- 60 -
for homocysteine, both vitamins, body mass, BMI, as well as several dietary intake
variables. One-way repeated measure ANOVA determined if the surgery altered
variables associated with the aims of this study. If a significant time interaction was
found, LSD was used as the Post-Hoc test for variables that showed differences across
time. Pearson product correlations were used to determine if significant relationships
existed between the variables. A p value of < 0.05 was deemed to be statistically
signficiant.
- 61 -
RESULTS
Data for nineteen female patients were analyzed for the current study. All subjects
underwent the same surgical procedure by the same surgeon. Patient characteristics are
displayed in Table 3. Mean age of participants was 45.7 (±8.8) years. Of the nineteen
participants, seventeen were Caucasian and two were African American. At baseline,
BMI ranged from 36.3 kg/m2 to 65.9 kg/m2 with a mean of 54.4 (±7.1) kg/m2. Minimum
and maximum measures of body mass at baseline were 98.3 kg to 193.9 kg with a mean
and standard deviation of 147.2 (±24.0) kg.
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TABLE 3. Baseline Patient Characteristics (mean, ±standard deviation):
Age (years) 45.4 (±8.8)
Body Mass (kg) 54.4 (±7.14)
BMI (kg/m2) 147.2 (±24.0)
Comorbidities (mean
number per patient)
4.6 (±1.6)
Comorbidities (%
prevalent)
Hypertension
Hyperlipidemia
CVD
Diabetes
Sleep Apnea
Depression
63%
42%
0%
42%
57%
57%
Medications (mean
number used per
patient)(% yes)CVD
(% yes)
1.8 (±1.5)
- 63 -
Following the surgical procedure, body mass was determined at 3 weeks, 3
months, 6 months, and 12 months. As shown in Figure 2, body mass was significantly
lower at each time point as compared to baseline, and at each subsequent time point
compared to the previous time. Body mass was reduced from 147.2 (±24.0) kg at baseline
to 135.0 (±21.6) kg at 3 weeks, 121.3 (±19.2) kg at 3 months, 106.3 (±16.2) kg at 6
months, and 97.0 (±17.0) kg after one year. Participants lost an average of 50.2 (±15.2)
kg over the course of the study. This is also demonstrated with percent change in mass
(Figure 3); percent change from baseline reached was 7.8 (± 1.5)% at 3 weeks, 18.0 (±
2.7)% at 3 months, 26.1 (± 4.0)% at 6 months and 33.9 (± 7.1)% at 12 months. Mean
BMI was reduced by 18.5 (4.7) kg/m2 at 12 months compared to baseline with a final
follow-up value of 35.9 (±6.2) kg/m2.
- 64 -
* indicates significantly different from all other visits
FIGURE 2.Change in Mass Between Baseline and One Year Follow up
VisitBaseline 3-Weeks 3-Months 6-Months 12-Months
Mas
s (K
g)
0
80
100
120
140
160
147.2
135.0
121.3
106.3
97.0
*
*
*
*
*
- 65 -
* indicates significantly different from all other visits
FIGURE 3. Percent Mass Loss Between Baseline and Follow-Up Visits
Visit
3 Weeks 3 Months 6 Months 12 Months
% M
ass L
oss
0
10
20
30
40
50
7.8
18.0
26.1
33.9
*
*
*
*
- 66 -
HOMOCYSTEINE
Because homocysteine data were not normally distributed, these data were
transformed using the natural log prior to comparison analysis. For ease in understanding,
non-transformed serum homocysteine levels are used in the text and in Figures 4 and 5.
Using repeated measures of variance analysis, no significant differences were observed
across time (p=0.879). Baseline homocysteine levels ranged from 7.2 μM/L to 22.8
μM/L with a mean of 10.4 (±3.5) μM/L. Mean values for homocysteine at the follow-up
time points were 11.7 (±3.9) μM/L at 3 weeks, 11.4 (±3.9) μM/L at 3 months, 11.6 (±2.9)
μM/L at 6 months, and 10.3 (±2.4) μM/L at 12 months. Serum levels ranged from 6.6
μM/L to 15.7 μM/L at the conclusion of the study. To show individual responses, each
participant’s values for the five visits are illustrated in Figure 5. As shown, the majority
of individuals had minimal changes with one participant having a tremendous drop across
time with others having transient increases followed by reductions to baseline
concentrations.
- 67 -
FIGURE 4.Mean Serum Homocysteine Levels
Visit
Baseline 3-Weeks 3-Months 6-Months 12-Months
HC
Y (m
icro
M/L
)
0
6
8
10
12
14
16
18
- 68 -
FIGURE 5Individual Serum Homocysteine Levels
Visit
HC
Y (m
icro
M/L
)
0
6
9
12
15
18
21
24
Baseline 3-Weeks 3-Months 6-Months 12-Months
- 69 -
SERUM FOLIC ACID
Serum levels of folic acid were determined at baseline and each of the follow-up
visits. Using repeated measures of variance analysis, no significant differences were
observed over the course of twelve months (p=0.827) (Figure 6). At baseline, mean
serum folic acid level was 24.6 (±11.8) ng/mL. Minimum serum level was 10.6 ng/ml
while the maximum was 50.6 ng/ml. Mean values for folic acid at the follow-up time
points were 26.5 (±10.1) ng//mL at 3 weeks, 23.1(±10.9) ng//mL at 3 months, 21.1 (±7.7)
μM/L at 6 months, and 28.4 (±11.8) ng//mL at 12 months. Serum levels ranged from 11.8
ng/ml to 48.7 ng/ml at the conclusion of the study. To show individual responses, each
participant’s values for the five visits are illustrated in Figure 7.
- 70 -
FIGURE 6Mean Serum Folic Acid Levels
Visit
FOL(
ng/
mL)
010
20
30
40
Baseline 3-Weeks 3-Months 6-Months 12-Months
- 71 -
FIGURE 7 Individual Serum Folic Acid Levels
Visit
FOL
(ng/
mL)
069
121518212427303336394245485154
Baseline 3-Weeks 3-Months 6-Months 12-Months
- 72 -
SERUM VITAMIN B-12
Because vitamin B-12 data were not normally distributed, these data were
transformed using the natural log prior to comparison analysis. For ease in understanding,
non-transformed vitamin B-12 levels are used throughout the text and in Figures 8 and 9.
Using repeated measures of variance analysis, no significant differences were observed
across time (p=0.377). At baseline, mean serum vitamin B-12 was 617.6 (±351.1) pg/ml.
Minimum serum level was 253.0 pg/ml while the maximum was 1881.0 pg/ml. Mean
values for vitamin B-12 at the follow-up time points were 1071.3 (±449.8) pg/ml at 3
weeks, 953.8 (±610.8) pg/ml at 3 months, 774.5 (±539.6) pg/ml at 6 months, and 749.8
(±766.6) pg/ml at 12 months. Serum levels ranged from 205.0 pg/ml to 3677.0 pg/ml at
the conclusion of the study. To show individual responses, each participant’s values for
the five visits are illustrated in Figure 9.
- 73 -
FIGURE 8Mean Serum Vitamin B-12 Levels
Visit
B-1
2 (p
g/m
L)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
Baseline 3-Weeks 3-Months 6-Months 12-Months
- 74 -
FIGURE 9Individual Serum Vitamin B-12 Levels
Visit
B-1
2 (p
g/m
L)
0
500
1000
1500
2000
2500
3000
3500
4000
Baseline 3-Weeks 3-Months 6-Months 12-Months
- 75 -
DIETARY RESULTS
Results from the dietary logs are shown in Table 4. Nutrients that have been
shown to affect homocysteine levels are presented. Energy intake was significantly less
following the surgery as compared to baseline. Intake of protein, methionine, folic acid,
vitamin B-12, alcohol, and caffeine did not significantly change during the course of the
study.
- 76 -
TABLE 4. Daily Nutrient Intake At Baseline and Follow-up Time Points (mean, ±S.D.)
Baseline 3 weeks 3 months 6 months 12 Months P-Value
Calories
(kcal)
2186.4 (±657)
639.6 (±148)
821.1 (±281)
1009.3 (±284)
1202.6 (±552)
>.001
Protein
(g)
91.3 (±22.5)
53.1 (±16.2)
58.6 (±24.3)
67.7 (±18.6)
68.8 (±26.0)
.314
Methionine
(mg)
2.1 (±.52)
1.1 (±.31)
1.1 (±.41)
1.5 (±.42)
1.4 (±.56)
.239
Vitamin B-
12 (μg)
6.1 (±3.7)
3.1 (±1.4)
2.7 (±2.7)
3.8 (±3.9)
4.4 (±2.8)
.377
Folic Acid
(μg)
465.4 (±299.9)
132.2 (±60.3)
151.6 (±62.3)
193.5 (±93.1)
324.2 (±185.2)
.281
Alcohol
(g)
.43 (±1.31)
.00 (±0.0)
.10 (±.39)
.19 (±.79)
.01 (±.008)
.385
Caffeine
(mg)
57.2 (±42.8)
6.6 (±13.8)
19.7 (±26.8)
31.6 (±36.2)
79.86 (±117.5)
.179
- 77 -
CORRELATIONS
Pearson-Product Correlations were performed between baseline values of
homocysteine and body mass, vitamin status, and other dietary factors that are known to
influence homocysteine concentrations. Correlation coefficients and p values from these
associations are shown in Table 5. Vitamins B-12 and folic acid were not significantly
correlated (p>0.05) to homocysteine at baseline with p-values of .097 and .148,
respectively. However, serum homocysteine was significantly correlated to serum B-12
and folic acid levels at the twelve month follow-up visit (r= -.486, p=.041 for B-12 and r=
-.522, p=.026 for folic acid). None of the other dietary variables (protein, methionine,
caffeine, and alcohol intake) were significantly correlated with homocysteine at either
time period. The total change in serum homocysteine over the course of the study was
significantly correlated to the overall change in serum B-12 (r= -.624, p=.006), serum
folic acid (r= -.511. p=.030), and BMI reduction (r= .580, p=.028). For vitamin B-12 and
folic acid, increased homocysteine levels were associated with decreased vitamin levels
in the blood. Decreased BMI was associated with increased homocysteine. The scatter
plot in Figure 10 displays the relationship between change in transformed homocysteine
and BMI reduction. Figures 11 and 12 display scatter plots for change in Ln HCY and
change in Folic Acid and change in Ln vitamin B-12, respectively.
- 78 -
TABLE 5. Pearson-Product Correlations
Baseline Ln Homocysteine
12 Month Ln Homocysteine
Δ Ln Homocysteine
Baseline BMI r= -.275, p=.270
Baseline Body Mass r= -.292, p=.239 Baseline Ln B-12 r = -.403, p=.097 Baseline FOL r = -.355, p=.148 Baseline Alcohol r = -.176, p=.546 Baseline Caffeine r = -.141, p=.631 Baseline Methionine r = -.468, p=.091 12 Month BMI r=.226, p=.367 12 Month Body Mass r=.150, p=.553 12 Month Ln B-12 r = -.486, p=.041 12 Month FOL r = -.522, p=.026 12 Month Alcohol r = -.116, p=.692 12 Month Caffeine r = -.150, p=.608 12 Month Methionine r = .244, p=.400 Δ Ln B-12 r = -.624, p=.006 Δ FOL r = -.511. p=.030 Δ Body Mass r = .460, p=.055 Δ BMI r = .580, p=.028 Δ Alcohol r = -.308, p=.285 Δ Caffeine r = -.241, p=.406 Δ Methionine r = -.259, p=.372
- 79 -
Figure 10 Correlation Between Δ Ln HCY and BMI Reduction
r = .580, p=.028
Δ Ln HCY
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
BM
I Red
uctio
n (K
g/m
2 )
10
12
14
16
18
20
22
24
26
28
30
- 80 -
FIGURE 11Correlation Between Δ HCY and Δ B-12
r = -.624, p=.006
Δ Ln HCY-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Δ Ln
B-1
2
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
- 81 -
FIGURE 12Correlation Between Δ Ln HCY and Δ Folic Acid
r = -.511. p=.030
Δ Ln HCY-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Δ F
olic
Aci
d
-40
-30
-20
-10
0
10
20
30
40
- 82 -
DISCUSSION
The primary aim of this study was to examine the change in serum homocysteine
levels following laparoscopic Roux-en-Y gastric bypass surgery in morbidly obese
patients. A secondary aim was to study the associations between concentrations of
homocysteine at baseline and 12 months following surgery, with vitamin B-12 and folic
acid, as well as anthropometric measures (body mass and body mass index) at the
respective time points. Because of the proven reduction in other cardiovascular disease
risk factors post surgery, as well as the prophylactic regimen of vitamin supplementation
that was prescribed to our subjects, we hypothesized that serum homocysteine would
decrease following surgery. A recent study also found surgery patients given a large daily
supplement of B-12 had decreased plasma homocysteine levels post gastric bypass
surgery. 217 Because vitamin B-12 and folic acid are involved in the metabolism of
homocysteine in the cell, we also hypothesized that serum homocysteine would be
negatively correlated with blood levels of vitamins B-12 and folic acid, with higher levels
of vitamins associated with lower concentrations of homocysteine.
Our results do not support our first hypothesis as homocysteine was not
significantly changed from baseline to twelve months post surgery (p=.879). Whereas
baseline values of homocysteine and folic acid and vitamin B-12 were not correlated,
there was a significant negative correlation between these markers at 12 months. These
latter correlations were expected as both folic acid and vitamin B-12 are involved in the
- 83 -
metabolism of homocysteine to methionine. Initially, it was surprising that correlations
were not apparent at baseline. However, upon further consideration, this lack of
association may be because at adequate levels of the vitamins, further reduction in
homocysteine is not going to be apparent. In other words, when B-12 and folic acid
reach a certain level, they may no longer influence homocysteine metabolism. It may be
that these vitamins only influence homocysteine levels when there is a shortage. As the
participants of this study were not considered deficient of either vitamin during the study,
it is not surprising that vitamin B-12 and folic acid were not found to be correlated with
homocysteine at most visits. Additionally, these data may indicate that other factors are
playing a role in homocysteine metabolism in this popuatlion. This may be related to
being in a condition of great metabolic fluctuations with tremendous reduction in dietary
intake (< 1,000 kcals per day for the first 6 months after surgery) and subsequent loss of
body weight. Although purely speculative, the dramatic loss in mass participants
experienced in initial months might have interfered with an aspect of the methionine or
folic acid cycles and affected the normal relationship between the vitamins and
homocysteine. Between visits at six and twelve months, mass loss was less dramatic than
between other visits and might partially explain why serum homocysteine correlated
stronger with serum vitamin B-12 and folic acid at twelve months.
While homocysteine, vitamin B-12, and Folate did not significantly change over
the course of the study, the individual responces were found to be negatively correlated
between change values. In other words, the more a participant’s vitamin status increased,
the more their homocysteine levls decreased (Figures 11 and 12).
- 84 -
As extreme obesity (BMI ≥ 40.0 kg/m2) has rapidly become more prevalent in
America, there has been a considerable rise in the number of patients having gastric
bypass surgery as treatment.180 While surgical treatment is considered successful with
over a 30% loss in body mass observed168, there are potential hazards, namely impaired
nutrient absorption that accompanies malabsorptive procedures, including RNYGB.
Many nutrients are preferentially absorbed in the proximal portion of the small intestine.
As this portion of the small intestine is bypassed in RNYGB, it is not surprising that
vitamin deficiencies are common following surgery. 168 Few studies have examined
plasma homocysteine levels after bypass surgery and those that have are inconclusive
with limitations on interpreting their findings. The primary results from several of these
earlier works show an increase in plasma homocysteine levels post surgery. 24,53, 94, 170
However, three studies did not appropriately address vitamin status in their analysis. 24,
94,170 In contrast, a more recent study using vitamin supplementation found plasma
homocysteine levels decreased from 10.2 μM/L to 8.4 μM/L one year post surgery. 217
As stated, vitamins B-12 and folic acid are well-known predictors of plasma
homocysteine levels and if deficiency in one or more of these nutrients occurs, it will
likely increase homocysteine levels. Failure to measure these crucial vitamins is a
confounding factor in interpreting the homocysteine data in the three studies. Dixon et al
53 found higher homocysteine in 293 patients after Lap-Band surgery (10.4 μM/L
increased to 11.0 μM/L). However, they did not find changes in plasma vitamin B-12
over the course of the study (376 pg/mL at baseline and 365 pg/mL at follow-up).
Furthermore, plasma folic acid was found to be no different one year post surgery (8.4
ng/mL to 8.6 ng/mL). These findings challenge the notion that an increase in plasma
- 85 -
homocysteine after surgery is directly attributable to vitamin deficiencies. Following the
surgery, Dixon et al report that patients were recommended to supplement their diet with
a vitamin supplementation. Researchers found that when comparing patients who took
the recommended supplements to those who did not, the non-users had significantly
higher plasma homocysteine levels. The authors suggested that when undergoing mass
loss, patients had an altered dose-response relationship between plasma homocysteine
and vitamins B-12 and folic acid.53 It appears as if serum levels of vitamins B-12 and
folic acid levels are important but may not completely control serum homocysteine levels.
It is possible that other factors seem to alter homocysteine levels post-surgery. In the
current study, we examined serum vitamin B-12 and folic acid levels in addition to
homocysteine in an attempt to gain a better understanding of their relationships.
Only one participant had a homocysteine level at baseline that would be classified
as hyperhomocysteinemic (22.8 μM/L), using the most commonly cutoff of 15 μM/L 8, 75,
142, 152, 191, 192, 197. If a lower reference range of 12 μM/L had been used, five participants
would have been classified as hyperhomocysteinemic at baseline. The distribution curve
at baseline exhibited the characteristic positive skew that has been described in the
literature review. At the twelve month follow-up visit, two participants were
hyperhomocysteinemic with serum levels above 15 μM/L and five participants had serum
levels over 12 μM/L. The distribution curve at this time point also exhibited a positive
skew. Both serum folic acid and serum vitamin B-12 status were in normal ranges for all
patients at baseline. At no time point during the study were any of the patients considered
deficient in either of the nutrients. Folic acid deficiency is normally defined by any serum
level less than 3 ng/mL. The lowest baseline serum level was 10.6 ng/ml. Folic acid
- 86 -
levels remained relatively consistent throughout the course of the study. Average serum
folic acid levels at twelve months were well above the minimum of 3 ng/mL at 28.4
ng/ml (±11.8). The lowest serum level of folic acid at follow up was 11.8 ng/mL. During
the course of the study, no participant fell below 10 ng/ml of serum folic acid. Using a
serum level of 150-200 pg/ml as the criteria for vitamin B-12 deficiency no patients were
considered deficient and only three participants had serum levels less than 300 pg/ml at
baseline. One participant was close to the 200 pg/ml cutoff at 205.0 pg/ml at the end of
the study. Only two participants had follow-up serum levels below 300 pg/ml. At no
point during the study did any patient have serum vitamin B-12 levels below 200 pg/ml.
As there have been reports in the literature that various dietary variables can
influence homocysteine, several of these variables were assessed in our analysis.
Methionine levels were not significantly correlated with homocysteine during this study.
The literature shows that methionine intake usually does not influence fasting plasma
homocysteine levels even though this sulfur containing amino acid is the immediate
precursor to homocysteine in the metabolic pathway. 10, 171,200 Since participants in this
study were in a fasted state during each blood draw, the results of this study appear to
confirm this finding. Since methionine is derived from dietary protein, as expected, no
significant correlations between protein consumption and homocysteine were found.
Both alcohol and caffeine have been shown to be strong determinants of plasma
homocysteine levels. In this study, neither substance was correlated with homocysteine.
Most of the literature consistently reports that these substances only influence
homocysteine levels in high quantities. Between 4 and 6 cups of coffee per day have been
shown to raise homocysteine levels while moderate (<4 cups) had no effect. 136,182
- 87 -
Alcohol has a similar effect with only high levels of consumption having an effect on
plasma homocysteine. 68 The intake of caffeine and alcohol observed in this study were
moderate to none, therefore it was not surprising there were no significant correlations
with these variables.
Another interesting relationship observed in this study is between change of
serum homocysteine levels and change in BMI and mass. We hypothesized that a
decrease in mass would be correlated with a corresponding decrease in homocysteine.
The data from this study indicate the opposite. Significant correlations were apparent
between change of serum homocysteine and changes in BMI and mass. The loss of more
body mass/BMI was related to greater increases in serum homocysteine levels (Figure
10).
A possible explanation for the relationship between BMI/body mass loss and
increased serum homocysteine levels might be attributable to changes in lean body mass.
It has been shown that patients receiving gastric bypass not only lose fat-mass, but in
most cases will lose a considerable amount of lean body mass as well. The loss in lean
body mass might affect serum homocysteine in multiple ways. Primarily, fat mass loss
far exceeds that of lean body mass loss post-surgery. Carey et al found that after six
months, fat mass accounted for 66.5% of the total mass lost, while LBM loss was
responsible for the other 33.5%.33 Wadstrom et al found similar results but also
calculated LBM to fat mass ratios at each time from post surgery for a year. They found
roughly a 1:1 ratio at baseline, but a 1.58 ratio after one year.206 These findings suggest
that while lean body mass is lost post-surgery, proportionately, it is actually gained.
While absolute lean body mass is lost, relative lean body mass is increased. Several
- 88 -
studies have found a strong correlation between plasma homocysteine and LBM.18, 51, 153 Dierkes et al explained this finding by suggesting that since creatine is directly related to
muscle mass, differences in creatine formation might be responsible for the differences in
the formation of homocysteine.51 Creatine is formed endogenously from methionine.
Creatinine is the metabolic by-product of creatine, and creatinine is excreted in
proportion to the amount of lean body mass. Therefore, according to Battezzati, a higher
relative protein or lean body mass would lead to higher levels of methionine and
homocysteine.18 If this is true, it might offer some explanation as to why body mass loss
was correlated with homocysteine gains in the current study, especially during times of
greatest mass loss.
The findings that serum vitamin B-12 and folic acid levels had stronger
correlation with homocysteine at baseline and twelve months might be related to the
significant correlations between changes in serum homocysteine and loss in body mass
over the first follow-up periods. It seems plausible that during times of the greatest mass
loss the “normal” metabolism of homocysteine might be disturbed. However, this is
purely speculative as enzyme activity and concentrations of intermediates in the
homocysteine pathway, such as methionine synthase (MS), cystathionine synthase (CBS),
methionine adenosyltansferase (MAT), and S-Adenosylhomocysteine hydrolase were
not obtained.
This study has several limitations, including sample size and homogeneity of the
population. There were only 19 women, with the majority being Caucasian, that were
part of these analyses. Another limitation was that only one blood sample was analyzed
per participant, at each time point. It has been shown that a single homocysteine sample
- 89 -
will only be within ±16.1% to ±18.4% of the individual’s homeostatic set point with a
95% probability.42, 69, 192 Measurement of multiple samples around a given time frame
provide a smaller variation as there is day-to-day variability for an individual. If three to
five homocysteine determinations were made around each time point, it has been shown
that the mean will have a coefficient of variation of ±9% and ±7%.42, 69, 192 It is important
to note that biological variation is relevant in interpreting the data from the current study.
Because there was only one measurement taken; there is greater chance that the
homocysteine level recorded is not the participant’s true serum level and more subject to
day to day variability. Additionally, the study did not include a nonsurgical, stable,
control group for comparisons. A control group would have provided information about
homocysteine levels in the morbidly obese that do not have surgery.
Interestingly, there was one participant out of nineteen that experienced a
significant drop in her serum homocysteine levels over the course of the study. Ths
participant also had the highest baseline level of homocysteine (22.8 μM/L). This
particular participant was the only subject that had a BMI of less than 40 kg/m2 at
baseline. Over twelve months, she lost close to 80% of her excess weight and finished the
study with a BMI of 25.0 kg/m2. This subject’s vitamin status improved with each visit
and could have also contributed to the decrease in serum homocysteine levels. While this
is purely speculative, gastric bypass might have a significant lowering action on
homocysteine levels in patients with hyperhomocysteinemic levels at baseline. It also
could be possible that a reduction from an obese state to a ‘normal’ mass level will
provoke a decrease in homocysteine. While this was only one patient, no conclusions can
be made about her individual response. A larger sample size with more
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hyperhomocysteinemic patients at baseline may have changed the results and might be a
future research interest.
While the surgery did not decrease serum homocysteine levels as we
hypothesized, homocysteine levels did not increase as they did in other studies in which
vitamin status went uncontrolled. The maintenance of homocysteine levels after surgery
in this study may be linked with maintaining adequate levels of vitamin B-12 and folic
acid.
As the incidence of morbid obesity continues to rise, gastric bypass surgery will
remain a viable treatment option for these individuals. Having a greater understanding of
metabolic changes that occur, especially as they relate to comorbidities of obesity,
including cardiovascular disease, is critical. Future research is needed to further
understand the alterations in the metabolism of homocysteine during periods of rapid
mass loss.
In conclusion, serum homocysteine levels did not significantly change during the
study. 95% of the participants had normal homocysteine levels at baseline and throughout
the course of the study and did not vary significantly. Change in homocysteine was
significantly negatively correlated to change in BMI as well as change in weight between
baseline values and practically all other visits. Results indicate that when vitamin status is
controlled, Roux-en-Y gastric bypass surgery will not significantly alter homocysteine
levels in morbidly obese patients.
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